Mature Embryos Research Articles

High-efficiency somatic embryogenesis and plant regeneration in brinjal (Solanum melongena L.) var. Mattu Gulla

ABSTRACT The organogenesis protocol was established for a unique brinjal (Solanum melongena L.) variety, “Mattu Gulla”, which has a distinct size, color and flavor. The objective of this study was to develop a reliable and efficient protocol for the initiation and maturation of somatic embryos into plantlets. The explants were cultured on Murashige and Skoog medium augmented with 2,4-dichlorophenoxyacetic acid (2,4-D) (1.0 mg/L) and benzyl aminopurine (BAP) (1.5 mg/L) for callus initiation. The developing calli (45-days old) were transferred onto an MS medium augmented with plant growth regulators to determine the embryogenic potential of the explants. The proportion of embryogenic callus was higher in hypocotyl-derived calli (HC) with indole butyric acid (IBA) (1.5 mg/L) and BAP (1.0 mg/L with 2,4-D and thidiazuron (TDZ) (1.0 mg/L) than in cotyledon and leaf-derived calli. The embryo initiation was recorded on the 23rd day after subculture from HC with the 2,4-D and TDZ (1.0 mg/L) combination, and germination was recorded. The somatic embryos developed from cotyledon-derived calli showed the highest number of plantlets and a significant percentage of ex vitro survival. In contrast, the lowest number of plantlets was noted from the embryos of hypocotyl-derived calli and ex vitro survival of plantlets. Under greenhouse conditions, the acclimatized plantlets thrived and produced fruits with viable seeds. The established protocol in this study should serve as a platform for large-scale somatic embryogenesis and micropropagation of plantlets. The somatic embryo-based manipulation should be exploited as a biotechnological tool in crop breeding and improving desired agronomic traits.

Oocyte maturity, oocyte fertilization and cleavage-stage embryo morphology are better in natural compared with high-dose gonadotrophin stimulated IVF cycles

Research questionDoes high-dose gonadotrophin stimulation have an effect on oocyte and early-stage embryo development? DesignThis was a retrospective study including 616 natural cycle IVF (NC-IVF) and 167 conventional IVF (cIVF) cycles. In total, 2110 oocytes were retrieved and analysed in fresh cycles. In NC-IVF, only human chorionic gonadotrophin was applied to trigger ovulation. In cIVF, antagonist protocols with daily 150–300 IU of human menopausal gonadotrophins were performed. The effect of gonadotrophins on oocyte and early-stage embryo development was analysed. Primary outcomes were the occurrence of mature (metaphase II) oocytes, zygotes and embryos with good morphology at the cleavage stage 2 days after oocyte retrieval. ResultsThe mature oocyte rate (number of mature oocytes/number of retrieved oocytes) was higher in NC-IVF than cIVF cycles (89% versus 82%, adjusted odds ratio [aOR] 1.79, P = 0.001), as was the zygote rate per oocyte retrieved (70% versus 58%, aOR 1.76, P = 0.001) and the zygote rate per mature oocyte (79% versus 71%, aOR 1.62, P = 0.001). The percentage of zygotes that developed into cleavage-stage embryos was no different. For the transferred embryos, the probability of having a good embryo morphology with four blastomeres and a fragmentation of <10% (score 0) in cleavage-stage embryos was found to be higher in NC-IVF (proportional aOR for four blastomeres 2.00, P < 0.001; aOR 1.87 for a fragmentation score of 0, P = 0.003). ConclusionsOocyte maturity, oocyte fertilization and morphology of the cleavage-stage embryo are affected by high-dose gonadotrophin stimulation in fresh IVF cycles.

The source, level, and balance of nitrogen during the somatic embryogenesis process drive cellular differentiation.

Since the discovery of somatic embryogenesis (SE), it has been evident that nitrogen (N) metabolism is essential during morphogenesis and cell differentiation. Usually, N is supplied to cultures in vitro in three forms, ammonium (NH4+), nitrate (NO3-), and amino N from amino acids (AAs). Although most plants prefer NO3- to NH4+, NH4+ is the primary form route to be assimilated. The balance of NO3- and NH4+ determines if the morphological differentiation process will produce embryos. That the N reduction of NO3- is needed for both embryo initiation and maturation is well-established in several models, such as carrot, tobacco, and rose. It is clear that N is indispensable for SE, but the mechanism that triggers the signal for embryo formation remains unknown. Here, we discuss recent studies that suggest an optimal endogenous concentration of auxin and cytokinin is closely related to N supply to plant tissue. From a molecular and biochemical perspective, we explain N's role in embryo formation, hypothesizing possible mechanisms that allow cellular differentiation by changing the nitrogen source.

Polyethylene glycol induced somatic embryogenesis and plant regeneration from cotyledons of Vigna radiata (L.) Wilczek

• Developed a high-frequency regeneration protocol via Polyethylene glycol (PEG)–induced somatic embryogenesis. • 3-day old cotyledonary explants used for the development of early-stage- somatic embryos. • Incorporation of ABA and proline was effective in the maturation of somatic embryos. • Regenerated plants are phenotypically identical to the mother plants. • Genetic fidelity analysis of in vitro regenerated Vigna radiata plants was reported newly. The results show that the in vitro regenerated plants are true-to-type with no somaclonal variation. • This protocol could be useful for genetic transformation studies as well as large-scale production strategies of true-to-type plants without any somoclonal variation. An efficient plantlet regeneration protocol of green gram [ Vigna radiata (L.) Wilczek] via somatic embryogenesis (SE) using Polyethylene glycol (PEG) is reported in this study. Embryogenic calli were induced using 3-day-old cotyledonary explants on MS medium supplemented with picloram (5 µM). Early-stage somatic embryos were generated by treating these 3-day-old cotyledonary explants or calli derived from those explants with osmolytes such as, sorbitol, mannitol and PEG, among which PEG was found to be the ideal. Early-stage embryoids were subsequently developed into torpedo and cotyledonary stage embryoids in liquid cultures supplemented with abscisic acid (5 μM), proline (130 μM). Furthermore, plantlet conversion was achieved in MS medium with 6-Benzylaminopurine (BAP) 1 μM + gibberellic acid (GA 3 ) 12 μM with a ca . 35% regeneration frequency. Plantlets were established under field conditions after the confirmation of their survival. The survival rate of plantlets post-transplantation was ca. 99%. The field-grown plants were confirmed to be phenotypically identical to the mother plant. According to RAPD profiles, in vitro regenerated plants were monomorphic and similar to the control/mother plants. The regeneration protocol developed in this study could be efficiently utilized for genetic transformation studies and large-scale production strategies of true-to-type plants without any somaclonal variation.

Time-lapse imaging and developmental competence of donkey eggs after ICSI: Effect of preovulatory follicular fluid during oocyte in vitro maturation

Equus members exhibit very divergent karyotype, genetic plasticity, and significant differences in their reproductive physiology. Despite the fact that somatic cell nuclear transfer and intracytoplasmic sperm injection (ICSI) has gained relevance in the last few years in horses, few reports have been published exploring ovum pick up (OPU) and in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) in donkeys. Yet, some donkey species and breeds are considered endangered, and these assisted-reproductive technologies could help to preserve the genetic of valuable individuals. In this study, we tested the hypothesis that supplementation with jenny preovulatory follicular fluid (PFF) during IVM could improve oocyte developmental competence in the donkey. For this, in vitro nuclear maturation rates, cumulus cell expansion, and embryo development after ICSI of donkey COCs matured in culture media supplemented with fetal bovine serum (FBS) or donkey PFF, with a known metabolomic profile, were assessed. Time-lapse imagining was performed after ICSI of horse and donkey oocytes. Eight OPU sessions were done in five jennies with an average recovery rate of 69.2% (n = 45 COCs). Although lower cumulus cells expansion was observed in oocytes of PFF group (P = 0.0010), no significant differences were described in nuclear maturation rates and preimplantation embryo development between groups. Donkey ICSI embryos showed similar morphokinetics to horse ICSI embryos. Our study shows that supplementing IVM media with FBS or donkey PFF supports nuclear maturation and early preimplantation embryo development after ICSI in donkeys. To our knowledge, the present study is the first report of ICSI, time-lapse imaging and in vitro blastocyst production in donkey.

Production of Nematicidal Pinosylvin Stilbenes in Cell Suspension Cultures of Pinus koraiensis by Fungal Elicitation.

Pinosylvin stilbenes are natural phenolic compounds found in the Pinaceae family and act as phytoalexins. Some pinosylvin stilbenes have strong nematicidal activity against pine wood nematodes (PWNs: Bursaphelenchus xylophilus). Here, we established the efficient production of two pinosylvin stilbenes, dihydropinosylvin monomethylether (DPME) and pinosylvin monomethylether (PME), by cell suspension culture of Pinus koraiensis after fungal elicitation. DPME and PME were found in small amounts (less than 40 µg/g DW) in the stem bark and leaves of P. koraiensis plants. Cell suspension cultures were established from the cultures of calli derived from mature zygotic embryos of P. koraiensis in 1/2 Litvay medium containing 2.2 μM 2,4-D and 2.2 μM BA. Two types of fungal elicitors, fungal cell extract (CE) and fungal medium filtrate (MF), were prepared from three species of fungi (Penicillium chrysogenum, P. pinophilum, and P. roquefortii). CE and MF treatments strongly stimulated the production of PME and DPME in cultured cells. The production of PME in suspension cells of P. chrysogenum, P. pinophilum, and P. roquefortii MF treatments after 3 days was 5734 µg/g DW, 4051 µg/g DW, and 6724 µg/g DW, respectively. Pinosylvin synthase (PkSTS) and pinosylvin O-methyltransferase (PkPMT) are key genes in DPME and PME biosynthesis. qPCR analysis revealed that the expression of the PkSTS and PkPMT in cultured cells was highly enhanced after fungal elicitor treatment. The cell extracts after MF treatment resulted in 92.5 ± 7.8% immobilization of the adult PWNs and 63.7 ± 3.5% immobilization of the juvenile PWNs within 24 h. However, control cell extracts without MF treatment showed 11.3 ± 1.4% nematicidal activity against adult PWNs. Our results suggest that pinosylvin stilbenes can be produced from the cell culture of P. koraiensis after fungal elicitor treatment and can be used as nematicidal compounds against PWNs.

Norway spruce somatic embryogenesis benefits from proliferation of embryogenic tissues on filter discs and cold storage of cotyledonary embryos.

Vegetative propagation opens opportunities for the multiplication of elite tree progeny for forest regeneration material. For conifers such as Norway spruce (Picea abies) the most efficient vegetative propagation method is seed multiplication through somatic embryogenesis. Efficient culture methods are needed for somatic embryogenesis to be commercially viable. Compared to culturing as clumps, filter disc cultures can improve the proliferation of embryogenic tissue (ET) due to more even spread and better developmental synchronization. In this study, ET proliferation on filter discs was compared to proliferation as clumps. The study comprised 28 genotypes in four trials. The benefits of adding a pre-maturation step and the selection of fresh ET for the subculture were evaluated. Pre-maturation on hormone-free media before maturation did not significantly improve embryo yield but improved greenhouse survival from 69% to 80%, although there was high variation between lines. Filter disc cultivation of ET did result in better growth than in clumps but was more dependent on ET selection and the amount of ET than the clump cultivation method. Filter proliferation also favors certain lines. Post-maturation storage can be used to change the storage compound composition of the produced mature embryos. The embryo storage compound profile was analyzed after post-maturation cold storage treatments of 0, 4, 8, 31, and 61 weeks and compared to that of the zygotic embryos. Cold storage made the storage compound profile of somatic embryos closer to that of zygotic embryos, especially regarding the raffinose family oligosaccharides and storage proteins. Sucrose, hexose, and starch content remained higher in somatic embryos even through cold storage. Prolonged storage appeared less beneficial for embryos, some of which then seemed to spontaneously enter the germination process.

The translation regulator Zar1l controls timing of meiosis in Xenopus oocytes.

Oocyte maturation and early embryo development occur in vertebrates in the near absence of transcription. Thus, sexual reproduction of vertebrates critically depends on the timely translation of mRNAs already stockpiled in the oocyte. Yet how translational activation of specific mRNAs is temporally coordinated is still incompletely understood. Here, we elucidate the function of Zar1l, a yet uncharacterized member of the Zar RNA-binding protein family, in Xenopus oocytes. Employing TRIM-Away, we demonstrate that loss of Zar1l accelerates hormone-induced meiotic resumption of Xenopus oocytes due to premature accumulation of the M-phase-promoting kinase cMos. We show that Zar1l is a constituent of a large ribonucleoparticle containing the translation repressor 4E-T and the central polyadenylation regulator CPEB1, and that it binds directly to the cMos mRNA. Partial, hormone-induced degradation of Zar1l liberates 4E-T from CPEB1, which weakens translational repression of mRNAs encoding cMos and likely additional M-phase-promoting factors. Thus, our study provides fundamental insights into the mechanisms that ensure temporally regulated translation of key cell cycle regulators during oocyte maturation, which is essential for sexual reproductivity.

Endosperm ontogeny through the lens of epigenetics

Endosperm tissue is crucial to seed development. Monocots and dicots have evolved different fates for their endosperm: it is usually consumed in dicots during seed development, while monocots seeds retain the endosperm till maturity. Recently, Wu et al. addressed the role of divergent epigenetic regulation over functionally conserved IKU2 gene contributing to silenced or persistent endosperm proliferation and has provided insights into diverged seed ontogeny pattern in plants. Here, we highlight the novel findings of this study and their potential significance. Seed production represents a remarkable life-history adaptation that has been established during plant evolution. In flowering plants, a double fertilization event initiates seed development, producing embryo and the endosperm. Endosperm (3n) is a triploid outcome of second fertilization, which occurs when a female central cell (2n) fuses with one of the two male gametes (n) carried by the pollen tube (Ingram, 2020). It develops precociously and is primed to perform nutritional and developmental functions for embryo growth (Povilus & Gehring, 2022). Different taxa have evolved specific seed formation patterns that elicit different levels of endosperm perseverance during development (Baroux et al., 2002). As a result of this, the endosperm-to-embryo ratio in mature seeds has varied gradually among different plants. As an example, endosperm in cereals is formed and retained as a nutritive reserve till seed maturity. In several eudicots such as Arabidopsis, early formed endosperm is substantially devoured to serve during embryo maturation. In peas with non-persistent endosperm, the tissue appears to be absorbed in the early free nuclear division phase before cell wall formation. The Podostomenaceae and Orchidaceae are the two endosperm-free lineages because their seeds lack or terminate endosperm nuclear divisions during early stages. Endosperm develops through two major phases: first syncytial involving continuous nuclear divisions, and the subsequent cellularization in which the free nuclear state develops cell wall (Baroux et al., 2002). Despite that the embryo produces the next generation progeny, endosperm proliferation and cellularization timing is crucial to embryo viability.

Functional Analysis of Glucose-6-Phosphate Translocator PoGPT1 in Embryogenic Callus Formation of Tree Peony

Tree peony (Paeonia ostii) is an ornamental flowering plant that is generally recalcitrant to establishment of a mature somatic embryo regeneration system in vitro. Glucose-6-phosphate translocator (GPT) plays an important regulatory role in embryogenesis of plants. In this study, PoGPT1 was cloned, and a bioinformatic analysis and functional verification of the gene were performed. The results showed that PoGPT1 encoded a polypeptide of 392 amino acids, which was a basic non-secreted hydrophobic transmembrane protein, and was mainly localized in the plastids. PoGPT1 was highly expressed in tree peony leaves, and its transcript abundance increased with the progression of zygotic embryo development. Overexpression of PoGPT1 caused up-regulation of leafy cotyledon 1 (PoLEC1), somatic embryogenesis receptor-like kinase (PoSERK), and agamous-like15 (PoAGL15) in tree peony callus. In addition, PoGPT1 overexpression promoted the increase in indole-3-acetic acid (IAA), 5-deoxystrigol (5DS), and brassinolide (BL) contents, especially of IAA, but reduced the contents of abscisic acid (ABA), 6-benzyladenosine (BARP), and 1-aminocyclopropanecarboxylic acid (ACC). The present research showed that PoGPT1 synergistically regulated the contents of endogenous hormones and expression levels of embryogenesis-related genes to promote the embryonic development of tree peony. The results provide theoretical and technical support for the establishment of a tree peony embryogenic callus formation and subsequent research on somatic embryogenesis.

Accumulated effects of factors determining plant development from somatic embryos of Abies nordmanniana and Abies bornmuelleriana.

Despite a much later inception of somatic embryogenesis (SE) propagation protocols for gymnosperms than for angiosperm species, SE is becoming increasingly important due to its applications for commercial forestry. For many conifers, there are however still major bottlenecks in the SE plant production process limiting the use of SE for forestry operations, Christmas tree production and research projects. In the present case study, the effects on plant growth from different cultural factors applied during the SE developmental process were studied in two conifer species of high value for Christmas tree production. Seven clones of Abies nordmanniana and two clones of Abies bornmuelleriana were included in the study. Accumulated effects from cultural treatments were recorded from the start of germination of mature embryos of different quality scores through development into plants in the third growing period. Experimental factors of the cultural treatments included were: germination temperature, germination time, light conditions, survival ex vitro and traits for plant growth and vitality. The results reveal that most of the studied experimental factors influenced plant growth during the first three years however their relative importance was different. Plant survival rate at end of the nursery stage was strongly impacted by germination temperature (p<0.001), initial embryo score (p=0.007), clone (p<0.001) and to a lesser extend week of germination (p=0.017). This case-study highlights and quantifies the strong interrelation between the developmental steps of somatic embryogenesis and show the importance of considering all cultural steps when optimizing SE plant production protocols.

Evaluation of the Maturity and Gene Expression of Sheep Oocytes and Embryos Cultured in Media Supplemented with Marjoram (Origanum vulgare) Extract.

This study aimed to evaluate the effects of marjoram extract on oocyte maturation and gene expression in sheep oocytes and embryos. The first experiment studied the effect of the extract as an antioxidant to improve the in vitro maturation media used for sheep oocytes; the oocytes were matured in a TCM199 medium supplemented with 1 or 10 µg/mL of marjoram extract or the control, 0 µg, for 24 hr. Then, the maturation was estimated, and the gene expression was measured by using qPCR. The second experiment studied the effect of the extract on the development of sheep embryos produced in vitro; the fertilized oocytes were cultured in a SOF medium supplemented with 1 or 10 µg/mL of marjoram extract or the control, 0 µg, for 7 days. Then, the gene expression was measured using qPCR. The results showed that the marjoram extract did not improve nuclear maturation or the blastocyst rate. There was a significant increase in the level of GDF-9 gene expression in mature oocytes in the treatment groups. An increase in the expression of BCL-2 and EGR-1 genes was observed for the blastocysts in the 10 µg/mL group. We concluded that the marjoram extract did not improve nuclear maturation, but it did affect the expression of some genes in sheep oocytes and embryos.

Polish Contribution to Global Research on Somatic Embryogenesis

AbstractSomatic embryogenesis (SE) is a complex process that begins with regaining totipotency in some somatic cells, proceeds through embryo development and maturation, and ends with the formation of a whole plant. Since the first publications on SE in 1958, this regeneration process has been applied to the in vitro propagation of many plant species and has led to the development of some specific model systems. SE has been used to expand our understanding of the cytomorphological, physiological, biochemical, and genetic processes that govern the earliest developmental events in the life of plants. This paper summarizes the achievements of Polish research groups working on SE systems established for several plants (gentians, the tree fernCyathea delgadiiSternb., and conifers) and three model species (Arabidopsis thaliana,Medicagosativa, andM. truncatula). SE systems have used a broad spectrum of experimental approaches involving genomic tools (transcriptomics, proteomics, and chromatin analyses), physiological methods which focus on phytohormones, and cytological techniques. Studies on the experimental models ofA. thalianaandMedicagospp. have resulted in the identification of new genetic and epigenetic elements of the complex regulatory network controlling embryogenic induction in plant somatic cells. The protocol developed for ferns has provided a unique and simple system for cytological analysis of early SE events that occur in a single cell of initial explants. Gentian embryogenic suspension cultures have successfully been used in broad biotechnological applications, including plant transformation, protoplast isolation, culture, and fusion. Systems described for coniferous species effectively produced many vigorous somatic seedlings and cost-efficient storage of genotypes during clonal field-testing. The research undertaken by Polish scientists has resulted in developing experimental systems that have enabled significant advances in SE knowledge.

Comparison of oocyte collection methods using caprine ovaries and in vitro embryo culture

The objectives of this study were to elucidate the efficiency of oocyte recovery through different techniques, grading of oocytes, and optimization of the caprine in vitro embryo production system. Ovaries were collected from the local slaughterhouse and classified based on the presence of a corpus luteum (CL). A total of 442 cumulus-oocyte complexes (COC) were retrieved from 178 ovaries aseptically through aspiration, slicing, and slashing techniques. Collected COC were categorized according to the compactness of cumulus cells around the oocyte and the homogeneous cytoplasm. In vitro maturation of graded COC was done for 27 h. In vitro fertilization (IVF) of the matured oocytes was performed using frozen semen. At 16 h of IVF, presumptive zygotes were in vitro cultured for 7 d. It was found that 31% of ovaries had a CL and 69% did not have a CL. The number of oocytes recovered per ovary (2.89 ± 0.22) and the percentage of excellent oocytes, grade A (29.84 ± 4.00%) and B (44.80 ± 2.32%), were significantly greater from the slashing technique than from the aspiration (2.16 ± 0.30 and 11.80 ± 0.61%, 30.80 ± 3.56%, respectively) and slicing technique (2.40 ± 0.15 and 22.62 ± 7.81%, 16.10 ± 1.29%, respectively). Results revealed a significantly greater ( P < 0.05) rate of grade A (61.14 ± 5.83%, 36.04 ± 0.32%, and 22.51 ± 3.07%) and grade B oocytes (23.31 ± 1.61%, 28.90 ± 7.47%, and 28.29 ± 5.04%) reached the metaphase II stage recovered from 3 methods (slashing, slicing and aspiration, respectively). The cleavage and blastocyst rates of presumptive zygotes were found to be 74.15 ± 6.80% and 37.50%, respectively. This study suggests that the slashing technique is more suitable for harvesting a greater number and superior quality of COC. The culture conditions used in this experiment were found to be optimal and efficiently supported in vitro maturation, IVF, and subsequent development of goat embryos to the blastocyst stage. Based on the results of this study, the culture conditions and slashing technique could be used in caprine in vitro embryo production and advanced biotechnology research.

ROLE OF GROWTH HORMONE SUPPLEMENTATION IN POOR OVARIAN RESERVE PATIENTS UNDERGOING IN-VITRO FERTILIZATION- A RANDOMIZED CONTROLLED TRIAL

Growth hormone co-treatment in poor ovarian reserve patients has been suggested to increase number of mature oocytes and embryos available for implantation, improving live birth rate. The aim of the study was to assess if cotreatment with growth hormone during ovarian stimulation improves outcomes during IVF cycles.

Flower Type Affects the Timing of Embryo Development in Native Primula Vulgaris

In this study, pin and thrum flowered wild primrose (Primula vulgaris= Syn: Primula acaulis) populations, which are naturally distributed in Black Sea Region in Türkiye, were cultivated and their pollen viability, pollen tube growth and embryo development stages were investigated. As a result of the study, pollen viability in pollen of the pin flower type was 83.10%, while the in vitro germination rate was 69.43%. The viability and in vitro germination rates of pollen of thrum flower type were 84.91% and 67.92%, respectively. As a result of the squash preparation examinations, it was observed that the pollen tubes of both types started to germinate on the stigma on the 1 DAP (Day after pollination). On 4 DAP, it was determined that pollen tubes penetrated to the ovule. Embryo developmental stages examinations showed that, zygote formation occurred on 6 DAP in the pin flower type; while on the 20 DAP, the embryo was in the heart stage and finally on the 40 DAP, it was seen that it formed a mature cotyledonary stage embryo. Endosperm in the thrum type started to develop faster than the pin type and formed on the 6 and 7 DAP. In the thrum flower type, the proembryo formed on 10 DAP, but embryo formation could only be seen on 30 DAP. In the ovary examinations performed on the 40 DAP, some embryos were in the cotyledon stage while some were still in the globular or heart stage. It was also determined that no embryos were found in some ovules.

Cypripedium subtropicum embryo development and cytokinin requirements for asymbiotic germination

BackgroundCypripedium subtropicum is a unique, endangered lady’s slipper orchid with evergreen leaves on non-dormant shoots that is native to southwestern China. This study documents the major developmental events in C. subtropicum seed development from fertilization to seed maturity, determines the optimum period for seed collection, and examines the cytokinin requirements for asymbiotic germination and protocorm survival.ResultsStructural studies revealed that embryo development proceeded after successful fertilization at 60 days after pollination (DAP). At 105 DAP, a globular embryo with the shrinking inner seed coat was observed, and seeds collected at this time point exhibited optimal germination. After 120 DAP, most seeds had a mature embryo within the capsule, and within the cells of the embryo proper, numerous proteins/lipid bodies were present as the main storage products. In addition, the inner seed coat had compressed into a thin layer that tightly enclosed the embryo, while the outer seed coat had progressively elongated, resulting in a hair-like appearance of the mature seed. Histochemical staining using Nile red and toluidine blue O (TBO) indicated that the lignified inner and outer seed coats may lead to coat-imposed dormancy. Seeds collected at this stage germinated poorly. Analyses of cytokinin preferences and optimal concentrations for germination and protocorm survival showed that both 6-(γ,γ-dimethylallylamino) purine (2iP) and 6-benzylaminopurine (BA) enhanced germination compared with the control, although higher concentrations of BA (4 and 8 μM) suppressed germination. The protocorm survival rate improved with increasing 2iP concentration.ConclusionsThis study provides a reproducible procedure for culturing immature seeds of C. subtropicum based on a defined time schedule of seed development. In addition, the cytokinin 2iP was shown to improve germination and protocorm survival. This study provides a scientific basis for seedling establishment through asymbiotic seed culture for further reintroduction efforts.

Cryopreservation of coconut plumule using droplet vitrification

In the present investigation, four types of explants from mature zygotic embryos of coconut, viz., whole upper cotyledonary region without haustorium, half of the upper cotyledonary region without haustorium, plumule with a portion of radicle and exclusively plumular tissue, were cultured in 12 different media combinations to find a suitable explant which could be regenerated after cryopreservation. Explants were pre-cultured in medium with 0.4 and 0.5 M sucrose for three days followed by dehydration in PVS3 solution for different durations on a sterile aluminum strip after treating with loading solution. Strips were treated with liquid nitrogen inside a cryoflask until bubbling stopped and quickly transferred to a cryovial and stored for a minimum period of 24 hours in liquid nitrogen. It was observed that plumule alone or with a small portion of outer tissue was ideal for fast in vitro growth and recovery of whole plantlets of coconut in a medium supplemented with NAA alone. Addition of glutamine (5 mg L-1), TDZ (1 mg L-1) and NAA (18 mg L-1) aided the vigorous growth of plantlets. In control, the survival rate ranged from 60 to 90 per cent in plumule pre-grown in media containing 0.5 M sucrose after dehydration with PVS3 for various durations, whereas it was 14 to 75 per cent in cryopreserved ones. Considering the high survival (75%) and regrowth (35%) of cryopreserved plumule in the present study, there is much scope for further improvement of the procedure to find the right combination of factors so as to enhance complete recovery of plantlets without much injury to plumules during cooling and rewarming.

Somatic embryogenesis and plant regeneration from transverse thin cell layers of adult peach palm (Bactris gasipaes) lateral offshoots.

In this work, we report a successful protocol to obtain in vitro peach palm (Bactris gasipaes Kunth) “Diamantes 10” plants through somatic embryogenesis from transverse thin cell layer (TCL) explants, dissected from three sections (basal, medial, and apical) of lateral offshoots of adult plants cultured on different concentrations of 4-amino-3,5,6-trichloropicolonic acid (picloram). After swelling and development of primary callus in all treatments, without any strong effect of explant origin or picloram concentration, it was possible to observe the formation of embryogenic structures and the exact point from where they developed. Browning was also observed and correlated to the induction treatments, although it was not an impairment for the production of embryogenic structures. Subsequent maturation and conversion of somatic embryos into plantlets allowed their acclimatization 17 months after culture initiation (ACI), which was quicker than previous reports with juvenile tissues (from embryos or seed-germinated plantlets). To the best of our knowledge, this is the first report on peach palm regeneration through somatic embryogenesis from TCL explants from adult plants and could constitute, after fine-tuning the acclimatization stage, a tool for mass clonal propagation of elite genotypes of this open-pollinated crop, as well as for the establishment of conservation strategies of in situ gene bank plant accessions endangered due to aging and other threats.

An Optimized Protocol for Seed Sterilization and Shoot Regeneration from Mature Embryo in Wheat (Triticum Aestivum L.) var. HD2967

In the present study, an efficient protocol for seed sterilization was developed. This protocol used a 1.0 % bavistin solution, which provides a protective response against fungal contamination. It was observed that there was no significant difference among the effective sterilization treatments in comparison to seed germination and seedling length under in vitro conditions. Furthermore, the embryo excised from bavistin-treated seeds showed improvement in callus induction and shot regeneration in var. HD2967. 0.1% HgCl2-SDS solution used for seed sterilization was also optimized with 2.5 to 3.0 minutes as the best-suited duration, providing a completely sterilized environment for the seeds for healthy and fast growth. In addition, plant growth regulators (PGRs) were further used to screen the most effective concentration for callus to shoot formation using mature embryos. Auxin concentration of 2.0 mg/l in MS agar media was quite effective for callus induction from embryo explant. For shoot regeneration (SI-7) combination is suitable for a maximum number of shoots per callus. This combination of 2, 4-D, Zeatin, CuSO4, and TDZ showed good regeneration in the wheat variety HD2967. The optimized protocol displayed 100% callus induction and up to 80-85% regeneration in HD2967, with a high reproducibility rate paving the way for regenerating plantlets from mature wheat embryos.