Progressive Aggregation of 16 kDa Gamma-Zein during Seed Maturation in Transgenic Arabidopsis thaliana

Prolamins constitute a unique class of seed storage proteins, present only in grasses. In the lumen of the endoplasmic reticulum (ER), prolamins form large, insoluble heteropolymers termed protein bodies (PB). In transgenic Arabidopsis (Arabidopsis thaliana) leaves, the major maize (Zea mays) prolamin, 27 kDa γ-zein (27γz), assembles into insoluble disulfide-linked polymers, as in maize endosperm, forming homotypic PB. The 16 kDa γ-zein (16γz), evolved from 27γz, instead forms disulfide-bonded dispersed electron-dense threads that enlarge the ER lumen without assembling into PB. We have investigated whether the peculiar features of 16γz are also maintained during transgenic seed development. We show that 16γz progressively changes its electron microscopy appearance during transgenic Arabidopsis embryo maturation, from dispersed threads to PB-like, compact structures. In mature seeds, 16γz and 27γz PBs appear very similar. However, when mature embryos are treated with a reducing agent, 27γz is fully solubilized, as expected, whereas 16γz remains largely insoluble also in reducing conditions and drives insolubilization of the ER chaperone BiP. These results indicate that 16γz expressed in the absence of the other zein partners forms aggregates in a storage tissue, strongly supporting the view that 16γz behaves as the unassembled subunit of a large heteropolymer, the PB, and could have evolved successfully only following the emergence of the much more structurally self-sufficient 27γz.

Aprecierea potențialului morfogenetic și regenerativ al genotipurilor de triticale în cultura in vitro

Triticale is an important cereal crop grown throughout the world. Research showed that the regen-eration of young plants from mature embryos triticale depended on genotypic characteristics. The fre-quency of callusogenesis varied depending on the genotype and was: 188 TR5027 - 80.19%, Ingen 93 standard - 92.02% and Ingen 35 - 98.45%, and the frequency of rhizogenesis compared to embryogenesis proved to be high and constituted on average 57.35%. Only in 34.53% of the morphogenic callus, the de-velopment was of the embryoid type. The average frequency of regeneration was 35.07%. The dispersive analysis of the obtained results shows a significant influence of the genotype in establishing a positive callusogenetic response (P <0.001), the influence power being 76.04%.

Transcriptomic Time-Series Analyses of Gene Expression Profile During Zygotic Embryo Development in Picea mongolica

Zygotic embryogenesis is a critical process during seed development in gymnosperms. However, knowledge on the genome-wide transcriptional activation that guides this process in conifers is limited, especially in Picea mongolica. This tree species is endemic to semiarid habitats of Inner Mongolia in China. To extend what is known about the molecular events underpinning its zygotic embryogenesis, comparative transcriptomic analyses of gene expression in zygotic embryos were performed by RNA sequencing in P. mongolica. Our results showed that most changes in transcript levels occurred in the early embryonic pattering determination and formation of mature embryos. Transcripts related to embryogenic competence, cell division pattern, hormones, and stress response genes were identified during embryogenesis. Auxin is essential for early embryo patterning and pre-cotyledon embryonic formation. However, ABA is a major regulator of embryo maturation. Moreover, we found that methylation-related gene expression is associated with activation of early-stage embryos, late embryogenesis abundant proteins, and storage/energy-related genes with late and mature embryos. Furthermore, network analysis revealed stage-specific and multistage gene expression clusters during embryogenesis. WOX, MYB, AP2, and HLH transcription factors seem more relevant to embryogenesis in different stages. Our results provide large-scale and comprehensive transcriptome data for embryo development in P. mongolica. These data will lay a foundation for the protection and utilization of P. mongolica resources.

Somatic embryogenesis in mature zygotic embryos of Picea pungens

This study developed somatic embryogenesis protocols for Picea pungens (Engelm), an important ornamental species, including initiation, proliferation, maturation, germination, and acclimation. Somatic embryogenic tissues were induced from mature zygotic embryos of five families, with a frequency of $$\ge $$ ≥ 22% for each. Embryogenic tissues (ET) from 13 clones of three families were proliferated for one week, achieving an average rate of 179.1%. The ET of 38 clones of three families were cultured in maturation medium for six weeks; 188 mature embryos on average were counted per gram ET cultured, of which $$\ge $$ ≥ 81.1% appeared normal, and each clone developed at least 28 normally matured embryos. A total of 69.9% or more of cotyledonary somatic embryos germinated normally and developed into normal emblings. The experiment of transplanting the emblings into a greenhouse had an average survival rate of 68.5%. Considerable variation among and within families during initiation and proliferation was observed, but this variation decreased in the maturation and germination. Changing the concentration of plant growth regulator of the initiation medium did not significantly change the initiation frequency. We recommend incorporating these protocols into the current Picea pungens practical programs, although further research is essential to increase efficiencies and reduce cost.

Cell Type Matters: Competence For Alkaloid Metabolism Differs In Two Seed-Derived Cell Strains of Catharanthus Roseus.

Since the discovery of the anticancer drugs vinblastine and vincristine, Catharanthus roseus has been intensively studied for biosynthesis of several terpene indole alkaloids (TIAs). Due to their low abundance in plant tissues at a simultaneously high demand, modes of production alternative to conventional extraction are mandatory. Plant cell fermentation might become one of these alternatives, yet decades of research have shown limited success to certain product classes, leading to the question, how to preserve the intrinsic ability to produce TIAs (metabolic competence) in cell culture? We used the strategy to use the developmental potency of mature embryos to generate such strains. Two cell strains (C1and C4) from seeds of Catharanthus roseus were found not only to differ morphologically, but also in their metabolic competence. This differential competence became manifest under phytohormone elicitation, but also upon feeding with alkaloid pathway precursors. The more active strain C4 formed larger cell aggregates and was endowed with longer mitochondria. These cellular features were accompanied by higher alkaloid accumulation in response to methyl jasmonate (MeJA) elicitation. The levels of catharanthine could be increased significantly, while the concurrent vindoline branch of the pathway was blocked, such that no bisindole alkaloids were detectable. By feeding vindoline to MeJA elicited C4 cells, vincristine became detectable; however, only to marginal amounts. In conclusion, these results show that cultured cells are not just “de-differentiated”, but can differ in metabolic competence. In addition to elicitation, and precursor feeding, the cellular properties of the “biomatter” are highly relevant for the success of plant cell fermentation.

In vitro mature embryo culture protocol of einkorn (Triticum monococcum ssp. monococum) and bread (Triticumaestivum) wheat under boron stress

Mature embryos of einkorn (Triticum monococcum ssp. monococcum) and bread (Triticum aestivum) wheat were used for callus induction on media containing four (0, 1, 2 and 4 mg L− 1) different doses of 2,4-D and dicamba supplemented with under five (0, 6.2, 12.4, 24.8, and 37.2 mg L− 1) different boron stresses. The obtained callus was transferred to culture media with three (0, 0.5, and 2 mg L− 1) different BAP doses with five boron stresses for regeneration. The heaviest callus weight in einkorn wheat was in culture media with 1 mg L− 1 dicamba and 6.2 mg L− 1 (3.71 ± 0.13 g). Bread wheat had the heaviest callus weight on culture media with 4 mg L− 1 dicamba and 12.4 mg L− 1 (3.46 ± 0.40 g). Callus diameters were observed as the highest in culture media with 1 mg L− 1 and 12.4 mg L− 1 (1.10 ± 0.31) for einkorn and 4 mg L− 1 dicamba with 6.2 mg L− 1 boron (1.22 ± 0.27 cm) for bread wheat. Regeneration capacity was highest in control group with 6.2 mg L− 1 for both wheat genotypes (einkorn, 71.33% ± 11.78 and bread, 65.33% ± 10.80). The highest plantlet numbers were in only 2 mg L− 1 BAP (2.92 ± 0.88) for einkorn wheat and 0.5 mg L− 1 BAP supplemented with 6.2 mg L− 1 boron (3.71 ± 1.12) for bread wheat. This indirect regeneration protocol using mature embryos of einkorn and bread wheat under boron stresses may be useful for wheat breeding studies.

Influence of selected biogenic amines on development and demographic parameters of a temperate population of Cinara (Cupressobium) cupressi (Hemiptera, Aphididae)

Cinara (Cupressobium) cupressi, the cypress aphid, is a serious pest of Cupressus spp. and other Cupressaceae, considered to be one of the world’s 100 worst invasive alien species. The potential influence of selected biogenic amines (tryptamine, tyramine, putrescine, and cadaverine) on development and demographic parameters of a temperate population of the cypress aphid was investigated under laboratory conditions. Within parthenogenetic generations, the use of biogenic amines resulted in a significant reduction in the total fecundity and the average daily fecundity both in the fundatrix (F0) and the fundatrigeniae (F1), whereas this influence in the F5 generation was insignificant. In all parthenogenetic generations treated with amines, the dissected female abdomen contained a greater number of mature embryos compared to control samples. Cadaverine caused marked changes in the body length of all viviparous generations studied and a significant reduction in the number of developing embryos, compared to the control sample. The use of biogenic amines did not significantly affect the number of eggs produced by oviparous females but had an impact on their lifespan. Our results indicate that biogenic amines application may disrupt feeding behavior and thus leads to reduced fecundity, consistent with the observation of reduced embryo production. Consequently, bioamines can be used as an alternative agents to control this important pest species.

Callus Induction and Regeneration from Germinating Mature Embryos of Wheat (Triticum aestivum L.)

A high-performance protocol for callus induction was devised using germinating mature embryos of two local wheat (Triticum aestivum L.) landraces as explant. The results showed that callus development from germinating embryos was rapid starting one day after culture with an induction rate 20 to 25% higher than those of soaked embryos. In addition, the mean rate of growth of callus developed from germinating embryos was 60 to 70% higher than those cultured from soaked embryos. This study also demonstrated a higher frequency of green spots formation(48 to 56%)on callus derived from germinating embryos compared to their soaked counterpart (24 to 28%), suggesting a better differentiation potential of callus cultures derived from germinating embryo. These findings indicate that germinating mature embryo is more suitable explant for wheat callus induction and regeneration than the soaked mature embryo commonly employed for wheat callus culture.

Somatic Embryogenesis and Plant Regeneration in Viola canescens Wall. Ex. Roxb.: An Endangered Himalayan Herb

Viola canescens Wall. ex. Roxb. is an important but threatened medicinal herb found at 1500–2400 m above mean sea level in the Himalayas. Overexploitation and habitat preference have put the plant under serious threat. Thus, the present study was undertaken to develop an efficient protocol for in vitro propagation via somatic embryogenesis. The results revealed that plant can be regenerated successfully through somatic embryogenesis using leaf derived calli. Regular subculturing of calli on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D)/indole-3-butyric acid (IBA)/kinetin (Kn) and varying combinations of 2,4-D+Kn induced somatic embryogenesis. The maximum average number of somatic embryos (SE) (19.15 ± 2.66) was induced on the medium with 0.15 + 0.05 mg L−1 of 2,4-D and Kn, respectively, and this medium was used as a control. To enhance somatic embryo induction, the control MS medium was supplemented with l-glutamine (200–400 mg L−1) and casein hydrolysate (1–4%). The maximum average number of SE (27.66 ± 2.67) and average mature SE (13.16 ± 3.48) were recorded on the medium having 2 % l-glutamine and 50 mg L−1 casein hydrolysate. The induced SE were asynchronous, so, to foster their maturation, the culture medium (free from growth regulators) was supplemented with abscisic acid (ABA) and silver nitrate (AgNO3). The maximum average number (35.96 ± 3.68) of mature SE was noticed on MS medium supplemented with 1.5 mg L−1 ABA. Mature embryos had two well-developed cotyledons and an elongated hypocotyl root axis. The development of SE into plantlets was significant for embryos matured on the medium with AgNO3 and ABA, with 86.67% and 83.33% conversion on the medium with 0.20 mg L−1 6-benzylaminopurine (BAP). The plantlets thus produced acclimatized in a growth chamber before being transferred to the field, which showed 89.89% survival. The plants were morphologically similar to the mother plant with successful flowering.

Development of an efficient regeneration system for mature bombarded calli of Moroccan durum wheat varieties

This study examined the effects of various environmental and genetic factors on callus induction and plant regeneration of bombarded calli from mature embryos of durum wheat using the biolistic method. In this study, three Moroccan durum wheat varieties ('Isly', 'Amria', 'Marouane') were cultivated on two induction media (IM1 and IM2) with different nitrogen sources and contents. After that, each variety cultured on both induction media was transferred in to two regeneration media (RM1 and RM2) with different phytohormones, whereas each variety distributed through four combinations of treatments: IM1RM1, IM1RM2, IM2RM1, and IM2RM2. A completely randomized design with five replications per treatment for each genotype was used. Parameters considered in this study were phytohormones, nitrogen source and its content, plant variety, and their interactions. The study found that variety, medium and variety × medium interactions have a statistically significant effect on callus induction and plantlets regeneration. Prior to bombardment, the maximum percentage of callus induction was obtained under IM1. Conversely, the callus survival rate was not affected by the induction media once bombarded for all three varieties. The induction media had a significant effect on all regeneration parameters (p < 0.01). The variety ‘Isly’ showed the best regeneration efficiency after bombardment, with nearly 80% of plantlets regenerated under IM1 and RM2 combination. These media can be used for genetic transformation of durum wheat