Improvement of Bioactive Polyphenol Accumulation in Callus of Salvia atropatana Bunge.

Callus cultures of the Iranian medicinal plant Salvia atropatana were initiated from three-week-old seedlings on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) and various cytokinins. Although all tested hormonal variants of the medium and explant enabled callus induction, the most promising growth was noted for N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU)-induced calli. Three lines obtained on this medium (cotyledon line-CL, hypocotyl line-HL, and root line-RL) were preselected for further studies. Phenolic compounds in the callus tissues were identified using UPLC-MS (ultra-performance liquid chromatography-mass spectrometry) and quantified with HPLC (high-performance liquid chromatography). All lines exhibited intensive growth and contained twelve phenolic acid derivatives, with rosmarinic acid predominating. The cotyledon-derived callus line displayed the highest growth index values and polyphenol content; this was exposed to different light-emitting diodes (LED) for improving biomass accumulation and secondary metabolite yield. Under LED treatments, all callus lines exhibited enhanced RA and total phenolic content compared to fluorescent light, with the highest levels observed for white (48.5-50.2 mg/g dry weight) and blue (51.4-53.9 mg/g dry weight) LEDs. The selected callus demonstrated strong antioxidant potential in vitro based on the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) tests. Our findings confirm that the S. atropatana callus system is suitable for enhanced rosmarinic acid production; the selected optimized culture provide high-quality plant-derived products.

Single versus multiple metabolite quantification of in vitro starch digestion: A comparison for the case of pulse cotyledon cells.

Different quantification methods for in vitro amylolysis were compared for individual chickpea and lentil cotyledon cells (ICC) as a relevant case study. For the first time, much-applied spectrophotometric methods relying on the quantification of certain functional groups (i.e., DNS, GOPOD) were compared to chromatographic quantification of starch metabolites (HPLC-ELSD). The estimated rate constant and linked initial rates of amylolysis were highly correlated for DNS, GOPOD, and HPLC-ELSD. However, absolute amylolysis levels depended on the applied method and sample-specific metabolite formation patterns. Multiresponse modelling was employed to further investigate HPLC-ELSD metabolite formation patterns. This delivered insight into the relative importance of different amylolysis reactions during in vitro digestion of pulse ICC, proving that maltotriose and maltose formation determined the overall amylolysis rate in this case. Multiresponse reaction rate constants of maltotriose and maltose formation were highly correlated to single response amylolysis rate constants (and initial rates) obtained for all three quantification methods.

Field Availability and Avoidance of Imidacloprid-Treated Soybean Seeds and Cotyledons by Birds.

Treated seeds and their cotyledons can present a toxicological risk to seed-eating birds. To assess whether avoidance behavior limits exposure and consequently the risk to birds, three fields were sown with soybeans. Half of the surface of each field was sown with seeds treated with 42 g/100 kg seed of insecticide imidacloprid (T plot, treated) and the other half with seeds without imidacloprid (C plot, control). Unburied seeds were surveyed in C and T plots at 12 and 48 h post-sowing. Damaged seedlings were surveyed in C and T plots at 12 days post-sowing. The abundance and richness of birds was surveyed at the field level (without distinguishing between C and T plots) before, during, and after sowing, and 12 days post-sowing. Unburied seed density was higher in the headlands of the T plots than in the C plots, but did not differ between 12 and 48 h. The damage to cotyledons of seedlings was 15.4% higher in C plots than in T plots. The abundance and richness/ha of birds that eat seeds and cotyledons were lower after sowing, indicating a deterrent effect on birds by sowing imidacloprid-treated seeds. Although the variation in seed density over time does not allow solid conclusions to be drawn about the avoidance of seeds treated by birds, the seedling results suggest an aversive effect of imidacloprid-treated soybeans on birds. The dominant species was the eared dove (Zenaida auriculata), whose risk of acute poisoning by imidacloprid in soybean seeds and cotyledons was low, according to its toxicity exposure ratio, foraged area of concern, and foraged time of concern. Environ Toxicol Chem 2023;42:1049-1060. © 2023 SETAC.

Solid-state fermentation alters the fate of red kidney bean protein during buccal and gastrointestinal digestion: Relationship with cotyledon cell wall integrity.

The relationship between legume cotyledon cell wall and macromolecular nutrient digestibility has attracted increased attention. In this study, the effect of solid-state fermentation by Rhizopus oligosporus RT-3 on the digestibility of red kidney bean protein and its relationship with cotyledon cell integrity were investigated. Buccal digestion and gastrointestinal digestion were performed to compare the fate of protein between unfermented (F0) and fermented samples. Results showed a remarkable disruption in cotyledon cell integrity at the late fermentation period, and it was accompanied by a possible migration/degradation of protein matrix. Buccal and gastrointestinal digestion barely affected cell wall integrity at F0 but notably disintegrated cell morphology at 29 h of fermentation (F29). As this fermentation time, gastrointestinal digestion resulted in higher contents of soluble proteins, peptides, and free amino acids by 1.4-, 1.8-, and 2.5-fold, respectively. Therefore, solid-state fermentation facilitated the structural breakdown of cotyledon cell walls, thereby further improving protein digestibility.

Avoidance of neonicotinoid-treated seeds and cotyledons by captive eared doves (Zenaida auriculata, Columbidae).

Farmland birds can be exposed to neonicotinoids through the ingestion of treated unburied seeds and cotyledons. The aim of this study was to evaluate the avoidance of sorghum with imidacloprid, clothianidin or thiamethoxam, soybean with imidacloprid, and soybean cotyledons with imidacloprid or thiamethoxam on eared doves (Zenaida auriculata). Doves were fed with test food (untreated and neonicotinoid-treated sorghum, soybean or soybean cotyledons) and maintenance food (seed mix) for 3-5 days to study the repellency (primary repellency and conditioned aversion) and anorexia caused by neonicotinoid-treated food, followed by a 7-day period on maintenance food to study the persistence of the anorexic effect after neonicotinoid exposure. Immediately afterward, the same doves were exposed to treated test food during a second period of 3-5 days to study the potential reinforcement of food avoidance. Finally, doves were fed with untreated test food to test the capacity of the pesticide to induce conditioned food aversion against untreated food in subsequent encounters. Intoxication signs and differences of body weight were determined. With sorghum, the three neonicotinoids produced a decrease in the consumption of treated seeds by >97% compared to control birds. However, this was not enough to prevent the death of 3/8 and 1/8 of the doves exposed to imidacloprid and clothianidin, respectively. Anorexia was clearly observed with neonicotinoid-treated sorghum. The birds did not avoid the untreated sorghum after exposure to the treated sorghum, indicating that avoidance is not generalized to the type of food without an associated sensory cue. The results obtained with soybean seeds and cotyledons were less conclusive because captive doves hardly consumed these foods, even without neonicotinoid treatment. The avoidance of sorghum seeds treated with neonicotinoids was insufficient to prevent poisoning and death of eared doves.

Intact cells: "Nutritional capsules" in plant foods.

Macronutrients of pulses or cereals are stored in the cotyledon or endosperm cells with protection from intact cell walls. However, pulses and cereals are generally processed into fine particles during food production. For example, after milling, the macronutrients enclosed in the intact cells are released and are easily accessible to digestive enzymes in the gastrointestinal tract, leading to high metabolic responses. Therefore, studies on the health effects of intact cells and developing an alternative ingredient with a higher proportion of intact cells are areas of emerging interest. In this review, we highlighted the smallest unit of whole grain, an individual cell, as "nutritional capsules" and elucidated the structure-function of the nutritional capsules, followed by isolation techniques, as a potential novel functional ingredient and food. The polysaccharides' monomeric composition, secondary structure, and interactions determine the cell wall properties including the cell detachment during isolation and isolated cell properties. The intact cellular structure is retained after mild food processing and digestion, thereby, contributing to a lower extent/rate of digestion of entrapped macronutrients. Furthermore, the excursed intact capsules in the colonic environment modulate the population and diversity of microbiota, favouring the increased production of the short-chain fatty acids (SCFAs). The structural schematic model of Type-I and Type-II cells is developed together with the schematics of the cell wall isolation process. The review provides a critical summary of the recent trends in intact plant cells as a functional-nutritional food. It paves the way for the industrial production of intact cells as a novel food ingredient.

Enzyme pre-milling treatments improved milling performance of chickpeas by targeting mechanisms of seed coat and cotyledon adhesion with various effects on dhal quality.

BACKGROUND: Dehulling and splitting are important elements of the milling process to produce dhal from pulses. However, grain that is difficult-to-mill because of tightly adhered seed coats or cotyledons that resist separation makes it difficult to achieve high quality dhal. Milling yields are reduced, energy inputs into the milling process are increased, and the resulting dhal can be of poorer quality, chipped or abraded. RESULTS: Eight enzyme pre-treatments were chosen based on the hypothesised mechanisms of seed coat and cotyledon adhesion established previously. Using a difficult-to-mill chickpea (Cicer arietinum L.) genotype, we examined the effects of these pre-treatments, over time, on laboratory-scale milling performance and dhal quality. We pioneered a texture analyser method to measure the flex of the cotyledons and the force required to cleave the cotyledons. The enzyme-induced changes ranged from negative (tough seed coat, weight loss, deleterious colour and texture, increased visual damage to cotyledons and increased kibble loss, concave cotyledons, increased flex, and changes in taste) to positive (brittle seed coat, increased seed volume, improved dehulling efficiency and splitting yield, reduced cotyledon cleavage force, and acceptable dhal quality and taste). CONCLUSION: All pre-treatments improved milling performance compared to milling the raw seed, although there was considerable variation between them. Two pre-treatments showed no improvement in milling yields compared to the water control, and several pre-treatments resulted in unacceptable qualities. Three pre-treatments, endo-polygalacturonanase, α-galactosidase and cellulase, show potential for commercial milling applications and could assist pulse millers globally to achieve high quality dhal at the same time as minimising milling effort. © 2021 Society of Chemical Industry.

Scarlet Flax Linum grandiflorum (L.) In Vitro Cultures as a New Source of Antioxidant and Anti-Inflammatory Lignans.

In vitro cultures of scarlet flax (Linum grandiflorum L.), an important ornamental flax, have been established as a new possible valuable resource of lignans and neolignans for antioxidant and anti-inflammatory applications. The callogenic potential at different concentrations of α-naphthalene acetic acid (NAA) and thidiazuron (TDZ), alone or in combinations, was evaluated using both L. grandiflorum hypocotyl and cotyledon explants. A higher callus induction frequency was observed on NAA than TDZ, especially for hypocotyl explants, with a maximum frequency (i.e., 95.2%) on 1.0 mg/L of NAA. The presence of NAA (1.0 mg/L) in conjunction with TDZ tended to increase the frequency of callogenesis relative to TDZ alone, but never reached the values observed with NAA alone, thereby indicating the lack of synergy between these two plant growth regulators (PGRs). Similarly, in terms of biomass, NAA was more effective than TDZ, with a maximum accumulation of biomass registered for medium supplemented with 1.0 mg/L of NAA using hypocotyls as initial explants (DW: 13.1 g). However, for biomass, a synergy between the two PGRs was observed, particularly for cotyledon-derived explants and for the lowest concentrations of TDZ. The influence of these two PGRs on callogenesis and biomass is discussed. The HPLC analysis confirmed the presence of lignans (secoisolariciresinol (SECO) and lariciresinol (LARI) and neolignan (dehydrodiconiferyl alcohol [DCA]) naturally accumulated in their glycoside forms. Furthermore, the antioxidant activities performed for both hypocotyl- and cotyledon-derived cultures were also found maximal (DPPH: 89.5%, FRAP 866: µM TEAC, ABTS: 456 µM TEAC) in hypocotyl-derived callus cultures as compared with callus obtained from cotyledon explants. Moreover, the anti-inflammatory activities revealed high inhibition (COX-1: 47.4% and COX-2: 51.1%) for extract of hypocotyl-derived callus cultures at 2.5 mg/L TDZ. The anti-inflammatory action against COX-1 and COX-2 was supported by the IC50 values. This report provides a viable approach for enhanced biomass accumulation and efficient production of (neo)lignans in L. grandiflorum callus cultures.

In vitro protein and starch digestion kinetics of individual chickpea cells: from static to more complex in vitro digestion approaches.

Attention has been given to more (semi-)dynamic in vitro digestion approaches ascertaining the consequences of dynamic in vivo aspects on in vitro digestion kinetics. As these often come with time and economical constraints, evaluating the consequence of stepwise increasing the complexity of static in vitro approaches using easy-to-handle digestion set-ups has been the center of our interest. Starting from the INFOGEST static in vitro protocol, we studied the influence of static gastric pH versus gradual gastric pH change (pH 6.3 to pH 2.5 in 2 h) on macronutrient digestion in individual cotyledon cells derived from chickpeas. Little effect on small intestinal proteolysis was observed comparing the applied digestion conditions. Contrary, the implementation of a gradual gastric pH change, with and without the addition of salivary α-amylase, altered starch digestion kinetics rates, and extents by 25%. The evaluation of starch and protein digestion, being co-embedded in cotyledon cells, did not only confirm but account for the interdependent digestion behavior. The insights generated in this study demonstrate the possibility of using a hypothesis-based approach to introduce dynamic factors to in vitro models while sticking to simple and cost-efficient set-ups.

Pea cell wall integrity controls the starch and protein digestion properties in the INFOGEST in vitro simulation.

The cell wall microstructure has been recognized to modulate the digestibility and bioaccessibility of nutrients in whole pulse foods, while the role of cell wall integrity is unclarified in the hydrolysis of intracellular nutrients during human gastrointestinal transit. Intact pea cells were isolated to prepare a series of cell wall integrity subjected to cooking and followed by the in vitro hydrolysis of starch and protein properties using the INFOGEST 2.0 in vitro simulation. Thermal properties showed that cell samples either in raw or cooked form with different wall integrity exhibited similar and higher starch gelatinization temperatures compared to the isolated starch counterpart. It was found that intact pea cells showed the limited hydrolysis extent of the maltose (16.2%) and NH2 (6.7%) compared to the damaged cells. In addition, intact cells also withheld the cell wall integrity throughout gastrointestinal digestion with minor rupture, and presented the higher protein molecular weight (70 kDa) in the SDS-PAGE profiles. Results suggested that the in vitro starch and protein digestion properties are modulated by the cell wall integrity, which may lead to lower glycemic response and open up the possibilities of designing health food products.

Phenotypic Study of Photomorphogenesis in Arabidopsis Seedlings.

Light is one of the most important environmental factors, serving as the energy source of photosynthesis and a cue for plant developmental programs, called photomorphogenesis. Here, we provide a standardized operation to measure physiological parameters of photomorphogenesis, including in hypocotyl length, cotyledon size, and anthocyanin content.

Pulse seeds as promising and sustainable source of ingredients with naturally bioencapsulated nutrients: Literature review and outlook.

Pulse seeds are nutritious and sustainable matrices with a high level of intrinsic microstructural complexity. They contain high-quality plant-based protein and substantial amounts of slowly digestible starch and dietary fiber. Starch and protein in pulses are located inside cotyledon cells that survive cooking and subsequent mechanical disintegration, hence preserving natural nutrient bioencapsulation. In this context, several authors have explored a number of techniques to isolate individual cotyledon cells from these seeds, aiming to unveil their digestive and physicochemical properties. In recent years, isolated pulse cotyledon cells are also being highlighted as promising novel ingredients that could improve the nutritional properties of traditionally consumed food products. Even more, they could enable to implement a strategy for increasing pulse intake in populations where these seeds have not been traditionally consumed. This review mainly focuses on the reported digestive, physicochemical, and technofunctional properties of pulse cotyledon cells isolated through different techniques, preceded by a descriptive summary of the nutritional properties, structural organization, and traditional process chain of pulse seeds. It also offers an outlook of research directions to take, based on the identified research gaps. All in all, it is clear that isolation of pulse cotyledon cells using diverse techniques constitutes a promising strategy for the development of pulse-based ingredients where natural bioencapsulation of macronutrients is preserved. However, much more research is needed at the level of ingredient characterization to better understand the effect of starting pulse seed material, isolation technique, and isolation conditions on the nutritional and functional properties of the finished product(s) where the isolated cells are (to be) used.

Structural and quantitative changes of starch in seed of Trigonella persica during germination.

Starch, the most abundant carbohydrate reserve in plant seeds, is synthesized and stored in the cotyledons of some plants. In seeds of Trigonella persica (Fabaceae), starch appears during germination and forms granules that are composed of amylose and/or amylopectin. In this study, both quantitative and qualitative traits of starch were evaluated during the germination of T. persica seed. The quantitative assay and microscopic imaging showed that there was a low-amylose starch in the cotyledons and its amount reached the highest at 48 h after imbibition (HAI). According to the SEM images of hydrogels, FT-IR spectra, and analysis of extracted starches, the purity of this starch was decreased during germination. So that the extracted starch from cotyledons of non-germinated seeds (ESCN), with the highest purity and the lowest protein content, was different from all the extracted starches. Therefore, it seems that ESCN was an amylopectin rich type starch, which had low amylose to amylopectin ratio and as waxy starch typically, it can have a cohesive and gummy texture compared with ordinary starch.

Effect of Heat Stress on Seed Protein Composition and Ultrastructure of Protein Storage Vacuoles in the Cotyledonary Parenchyma Cells of Soybean Genotypes That Are Either Tolerant or Sensitive to Elevated Temperatures.

High growth temperatures negatively affect soybean (Glycine max (L.) Merr) yields and seed quality. Soybean plants, heat stressed during seed development, produce seed that exhibit wrinkling, discoloration, poor seed germination, and have an increased potential for incidence of pathogen infection and an overall decrease in economic value. Soybean breeders have identified a heat stress tolerant exotic landrace genotype, which has been used in traditional hybridization to generate experimental genotypes, with improved seed yield and heat tolerance. Here, we have investigated the seed protein composition and ultrastructure of cotyledonary parenchyma cells of soybean genotypes that are either susceptible or tolerant to high growth temperatures. Biochemical analyses of seed proteins isolated from heat-tolerant and heat-sensitive genotypes produced under 28/22 °C (control), 36/24 °C (moderate), and 42/26 °C (extreme) day/night temperatures revealed that the accumulation in soybean seeds of lipoxygenase, the ß-subunit of ß-conglycinin, sucrose binding protein and Bowman-Birk protease inhibitor were negatively impacted by extreme heat stress in both genotypes, but these effects were less pronounced in the heat-tolerant genotype. Western blot analysis showed elevated accumulation of heat shock proteins (HSP70 and HSP17.6) in both lines in response to elevated temperatures during seed fill. Transmission electron microscopy showed that heat stress caused dramatic structural changes in the storage parenchyma cells. Extreme heat stress disrupted the structure and the membrane integrity of protein storage vacuoles, organelles that accumulate seed storage proteins. The detachment of the plasma membrane from the cell wall (plasmolysis) was commonly observed in the cells of the sensitive line. In contrast, these structural changes were less pronounced in the tolerant genotype, even under extreme heat stress, cells, for the most part, retained their structural integrity. The results of our study demonstrate the contrasting effects of heat stress on the seed protein composition and ultrastructural alterations that contribute to the tolerant genotype's ability to tolerate high temperatures during seed development.

Insights into the starch gelatinization behavior inside intact cotyledon cells.

In this work, detailed structural changes of starch within intact cotyledon cells during differential scanning calorimetry (DSC) heating (water: cells ratio of 4:1, v/w) were investigated. Intact cotyledon cells containing raw starch granules from three legumes were isolated and used as materials, followed by simulate DSC heating up to different designated temperatures based on those gelatinization profiles of cells. The swelling power, solubility and gelatinization transition parameters of raw cells were significantly lower than pure starches. Upon simulate heating, all the starches inside intact cells were considered to maintain more amounts of crystalline and double-helix structures than pure starch counterparts. Meanwhile, the starch granules were not completely disrupted even heating up to 15 °C above conclusion temperature (Tc + 15 °C) for intact cells. The results showed clearly that the presence of intact cell wall exerts significant retarding or restricting effects on the process of starch gelatinization.

A cyclic dipeptide from the Chilean hazelnut cotyledons (Gevuina avellana Mol., Proteaceae).

The Chilean hazelnut (Gevuina avellana Mol., Proteaceae) is a southern South American nut consumed as a snack and included in different preparations of traditional Chilean cuisine. Recently we described the fatty acid profile, oxylipins, phenolic compounds, as well as the antioxidant capacity. The main compounds of the phenolic-enriched extract were only tentatively identified by spectrometric means. In the present work, we describe the isolation and full characterization of a cyclic dipeptide cyclo(Arg-Trp) and other compounds from the phenolic enriched extracts of the G. avellana cotyledons. Compounds were isolated by means of counter-current chromatography and structures were established by spectroscopic and spectrometric methods. This is the first report on small peptides in G. avellana and adds evidence on the possible beneficial effects of this nut in human health.

Quantitative proteomic, physiological and biochemical analysis of cotyledon, embryo, leaf and pod reveals the effects of high temperature and humidity stress on seed vigor formation in soybean.

BACKGROUND: Soybean developing seed is susceptible to high temperature and humidity (HTH) stress in the field, resulting in vigor reduction. Actually, the HTH in the field during soybean seed growth and development would also stress the whole plant, especially on leaf and pod, which in turn affect seed growth and development as well as vigor formation through nutrient supply and protection. RESULTS: In the present study, using a pair of pre-harvest seed deterioration-sensitive and -resistant cultivars Ningzhen No. 1 and Xiangdou No. 3, the comprehensive effects of HTH stress on seed vigor formation during physiological maturity were investigated by analyzing cotyledon, embryo, leaf, and pod at the levels of protein, ultrastructure, and physiology and biochemistry. There were 247, 179, and 517 differentially abundant proteins (DAPs) identified in cotyledon, embryo, and leaf of cv. Xiangdou No. 3 under HTH stress, while 235, 366, and 479 DAPs were identified in cotyledon, embryo, and leaf of cv. Ningzhen No. 1. Moreover, 120, 144, and 438 DAPs between the two cultivars were identified in cotyledon, embryo, and leaf under HTH stress, respectively. Moreover, 120, 144, and 438 DAPs between the two cultivars were identified in cotyledon, embryo, and leaf under HTH stress, respectively. Most of the DAPs identified were found to be involved in major metabolic pathways and cellular processes, including signal transduction, tricarboxylic acid cycle, fatty acid metabolism, photosynthesis, protein processing, folding and assembly, protein biosynthesis or degradation, plant-pathogen interaction, starch and sucrose metabolism, and oxidative stress response. The HTH stress had less negative effects on metabolic pathways, cell ultrastructure, and physiology and biochemistry in the four organs of Xiangdou No. 3 than in those of Ningzhen No. 1, leading to produce higher vigor seeds in the former. CONCLUSION: High seed vigor formation is enhanced by increasing protein biosynthesis and nutrient storage in cotyledon, stronger stability and viability in embryo, more powerful photosynthetic capacity and nutrient supply in leaf, and stronger protection in pod under HTH stress. These results provide comprehensive characteristics of leaf, pod and seed (cotyledon and embryo) under HTH stress, and some of them can be used as selection index in high seed vigor breeding program in soybean.

Starch digestion in intact pulse cotyledon cells depends on the extent of thermal treatment.

Starch digestion in pulse cellular matrices is primarily determined by the hindrance of cell walls limiting enzyme diffusion as well as the retention of starch granular structure. However, the effect of hydrothermal treatment on structure and digestion properties of entrapped pulse starches is not fully elucidated. In present study, we reported the variations in structure and enzyme susceptibility of pulse cells isolated at 60 °C followed by heated at 70, 80, 90, 100 °C, which were higher than the starch gelatinization temperature. Based on the thermal and crystalline properties, entrapped starches in pulse cells were not fully gelatinized even treated at 100 °C. Whilst, the digestion of entrapped pulse starches increased with higher temperature, but still much lower than the isolated starch treated at the same temperature. In addition to physical barriers (intact cell wall) and starch structural features (partial ordered crystalline structure), the soluble/insoluble proteinaceous materials in cells also synergistically reduced the starch digestibility.

Hormonal and gene dynamics in de novo shoot meristem formation during adventitious caulogenesis in cotyledons of Pinus pinea.

KEY MESSAGE: Several members of WOX and KNOX gene families and several plant growth regulators, basically cytokinins and auxins, play a key role during adventitious caulogenesis in the conifer Pinus pinea. Similar to Arabidopsis thaliana, Pinus pinea shoot organogenesis is a multistep process. However, there are key differences between both species, which may alter the underlying physiological and genetic programs. It is unknown if the genic expression models during angiosperm development may be applicable to conifers. In this work, an analysis of the endogenous content of different plant growth regulators and the expression of genes putatively involved in adventitious caulogenesis in P. pinea cotyledons was conducted. A multivariate analysis of both datasets was also realized through partial least squares regression and principal component analysis to obtain an integral vision of the mechanisms involved in caulogenesis in P. pinea. Analyses show that cotyledons cultured in the presence of benzyladenine during long times (2-6 days) cluster separately from the rest of the samples, suggesting that the benzyladenine increase observed during the first hours of culture is sufficient to trigger the caulogenic response through the activation of specific developmental programs. In particular, the most relevant factors involved in this process are the cytokinins trans-zeatin, dihydrozeatin, trans-zeatin riboside and isopentenyl adenosine; the auxin indoleacetic acid; and the genes PpWUS, PpWOX5, PpKN2, PpKN3 and PipiRR1. WUS is functional in pines and has an important role in caulogenesis. Interestingly, WOX5 also seems to participate in the process, although its specific role has not been determined.

Constitutive and induced insect resistance in RNAi-mediated ultra-low gossypol cottonseed cotton.

BACKGROUND: Besides fibers, cotton plants also produce a large amount of seeds with a high oil and protein content. The use of these seeds is restricted by their high contents of the terpenoid gossypol, which is harmful to humans and livestock. Using a genetic engineering approach, "Ultra-low gossypol cottonseed" (ULGCS) plants were produced by knocking down an enzyme that catalyzes the formation of a precursor of gossypol. This was accomplished via RNAi-mediated silencing of the target gene using a seed-specific α-globulin promotor. Since gossypol is also a crucial defense mechanism against leaf-feeding herbivores, ULGCS plants might possess lower herbivore resistance than non-engineered plants. Therefore, we tested the constitutive and inducible direct insect resistance of two ULGCS cotton lines against the African cotton leafworm, Spodoptera littoralis. RESULT: The herbivore was equally affected by both ULGCS lines and the control (Coker 312) line when feeding on fully expanded true leaves from undamaged plants and plants induced by jasmonic acid. When plants were induced by caterpillar-damage, however, S. littoralis larvae performed better on the ULGCS plants. Terpenoid analyses revealed that the ULGCS lines were equally inducible as the control plants. Levels of terpenoids were always lower in one of the two lines. In the case of cotyledons, caterpillars performed better on ULGCS cotton than on conventional cotton. This was likely caused by reduced levels of gossypol in ULGCS cotyledons. CONCLUSION: Despite those effects, the insect resistance of ULGSC cotton can be considered as largely intact and the plants may, therefore, be an interesting alternative to conventional cotton varieties.