Palm Tissue Research Articles

Cryopreservation of date palm tissue cultures by vitrification and assessment of genetic stability using ISSR analysis

In this work, a successful cryopreservation method of date palm tissue cultures by vitrification was established. Callus and somatic embryos of three date palm cultivars were exposed to dimethylsulfoxide (DMSO) and Plant Vitrification Solution 2 (PVS2) for different durations before plunging into liquid nitrogen for cryoprotection. After freezing phase, the cryopreserved cultures were thawed and cultured on recovery media. Among different times of exposure to DMSO (10%), 60 min gave best results of survival and regrowth of the date palm tissue cultures. Regarding recovery, ‘Zaghlool’ cultivar gave the highest values of callus fresh mass (0.5 g) and growth value (1.00). Also, the highest differentiation percentages (75%) and number of proliferated shootlets (8.50) were recorded with ‘Zaghlool’ cultivar. Concerning PVS2, survival and regrowth gradually increased as exposure time increasing till 30 min and then decreased. Unlike of callus cultures, the survival and regrowth rates of somatic embryos were relatively higher at all PVS2 exposure time. With regard to recovery, the genotype effect took same trend in cryopreservation of callus cultures, since ‘Zaghlool’ was superior in growth parameters in comparison to the other two cultivars. On the other hand, genetic stability of the cryopreserved date palm tissue cultures was assessed using ISSR markers. Among four screened primers, three gave monomorphic bands; all of these banding profiles were similar to those of non-cryopreserved cultures. Generally, the results of ISSR showed high genetic similarity suggesting that cryopreservation using vitrification does not affect genetic stability of date palm tissue cultures. The results of this study demonstrated PVS2 was more effective in cryoprotection of date palm tissue cultures compared with DMSO and cryopreservation by vitrification is a suitable tool for conservation of genetic stable date palm germplasm.

GWAS determined genetic loci associated with callus induction in oil palm tissue culture.

GWAS identified six loci at 25kb downstream of WAK2, a crucial gene for cell wall and callus formation, enabling development of a SNP marker for enhanced callus induction potential. Efficient callus induction is vital for successful oil palm tissue culture, yet identifying genomic loci and markers for early detection of genotypes with high potential of callus induction remains unclear. In this study, immature male inflorescences from 198 oil palm accessions (dura, tenera and pisifera) were used as explants for tissue culture. Callus induction rates were collected at one-, two- and three-months after inoculation (C1, C2 and C3) as phenotypes. Resequencing generated 11,475,258 high quality single nucleotide polymorphisms (SNPs) as genotypes. GWAS was then performed, and correlation analysis revealed a positive association of C1 with both C2 (R = 0.81) and C3 (R = 0.50), indicating that C1 could be used as the major phenotype for callus induction rate. Therefore, only significant SNPs (P ≤ 0.05) in C1 were identified to develop markers for screening individuals with high potential of callus induction. Among 21 significant SNPs in C1, LD block analysis revealed six SNPs on chromosome 12 (Chr12) potentially linked to callus formation. Subsequently, 13 SNP markers were identified from these loci and electrophoresis results showed that marker C-12 at locus Chr12_12704856 can be used effectively to distinguish the GG allele, which showed the highest probability (69%) of callus induction. Furthermore, a rapid SNP variant detection method without electrophoresis was established via qPCR-based melting curve analysis. Our findings facilitated marker-assisted selection for specific palms with high potential of callus induction using immature male inflorescence as explant, aiding ortet palm selection in oil palm tissue culture.

Towards the fourth decade of IOPRI’s oil palm clones: Upcoming new variety

Experience of IOPRI on oil palm tissue culture would be four decades in 2024. The first decade was theperiod of oil palm clone development and field testing. The second decade was a setback for oil palm research because of a high percentage of oil palm clone abnormalities in the field. Moving on from these experiences, tissueculture systems such as: including improving the protocol, controlling the duration of callus induction, delimitating the number of subcultures, and building of oil palm clones database, were managed to be improved. In this way, alow abnormality level is under control and each clone is traceable. It is also noted that the oil palm clone provides20-30% higher production than that derived from DxP crossing seed due to the more uniform growth of generatedplants. Besides the protocol, ortet selection is also key to improvement. Ortets are elite plants from selected progeny testing or re-clone of the progeny testing. Molecular analysis is applied as a control of genetic variation possibility, which may occur in a particular phase due to specific stimuli. However, until the present time, a clonevariety has not been released yet. Therefore, in the fourth decade, mass production is expected and oil palm clonevarieties will officially be released and commercialized

Identification of oil palm tissue culture contaminants by using DNA barcodes: preliminary results

The success of oil palm tissue culture is constrained by the microbes contaminating cultures in the laboratory. Accurate identification of contaminant species leads to the right strategies for solving the contamination problem. This research was done to identify contaminant species at the Indonesian Oil Palm Research Institute Tissue Culture Laboratory at its stages by using DNA barcodes. Bacteria identification was done by 16S rRNA sequencing, while fungi identification was done by ITS region sequencing. Preliminary results showed that bacteria and fungi were identified in the laboratory’s stages of the tissue culture process. Four species were identified in the dark room, two in the light room, and four in the advanced light room. The identified bacteria were Bacillus subtilis, Streptococcus parasuis, Priestia megaterium, and Bacillus sp. The identified fungi were Tritirachium candoliense, Penicillium citrinum, Irpex laceratus, Aspergillus reticulatus, and Perenniporia tephropora. More data is needed to determine the right strategies to control the contamination problem.

Quantification of DNA Methylation by ELISA in Epigenetic Studies in Plant Tissue Culture: A Theoretical-Practical Guide.

This chapter describes a step-by-step protocol for rapid serological quantification of global DNA methylation by enzyme-linked immunosorbent assay (ELISA) in plant tissue culture specimens. As a case study model, we used the coconut palm (Cocos nucifera), from which plumules were subjected to somatic embryogenesis followed by embryogenic calli multiplication. DNA methylation is one of the most common epigenetic markers in the regulation of gene expression. DNA methylation is generally associated with non-expressed genes, that is, gene silencing under certain conditions, and the degree of DNA methylation can be used as a marker of various physiological processes, both in plants and in animal cells. Methylation consists of adding a methyl radical to carbon 5 of the DNA cytosine base. Herein, the global DNA methylation was quantified by ELISA with antibodies against methylated cytosines using a commercial kit (Zymo-Research™). The method allowed the detection of methylation in total DNA extracts from coconut palm embryogenic calli (arising from somatic embryogenesis) cultivated in liquid or solid media by using antibodies against methylated cytosines and enzymatic development with a colorimetric substrate. Control samplesof commercially provided Escherichia coli bacterial DNA with previously known methylation percentages were included in the ELISA testto construct an experimental methylation standard curve. The logarithmic regression of this E. coli standard curve allowed methylation quantification in coconut palm samples. The present ELISA methodology, applied to coconut palm tissue culture specimens, is promising for use in other plant species and botanical families. This chapter is presented in a suitable format for use as a step-by-step laboratory procedure manual, with theoretical introduction information, which makes it easy to apply the protocol in samples of any biological nature to evaluate DNA global methylation associated with any physiological process.

Translocation and survival of trunk injected Beauveria bassiana (Hypocreales: Cordycipitaceae) in healthy date palm trees

ObjectiveThe date palm, Phoenix dactylifera L., is an ancient and valuable tree that provides food and other products. The date palm trees are attacked by several pests, including the red palm weevil, which is devastating to date palm plantations. Knowledge of the functionality of entomopathogenic fungi, including Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordycipitaceae) species, in the tree trunk is critical for controlling date palm weevil and other pests. The goal of this study was to assess the movement of the entomopathogenic fungus, B. bassiana, within the date palm tree. MethodsBeauveria bassiana (BbSA-4) mixed with food colors was trunk-injected using a balloon injector into healthy date palm plants. Trunks were cut into one-meter logs 2, 20, and 86 days post-injection. Each log was further dissected into four quarters to examine the present of the fungus. The appearance of food colors and the detection of fungal spores at different heights from the point of injection revealed the translocation of B. bassiana within the trunk. The samples were taken from several locations where food color could be visible, and the distance traveled by the fungal spores was measured. ResultsThe injected palm tissue samples were cultured on PDA media in the laboratory, and the present of fungal spores was confirmed. B. bassiana (BbSA-4) was found to be surviving in all treated date palm trees. The survival rate of isolate BbSA-4 averaged 70.5%, 34.9%, and 13.9% from dissected trunks examined at 2, 20, and 86 days, respectively, after injection. Isolate BbSA-4 was more apparent in the trunk after spiral injected than bottom injection. The findings revealed that using an entomopathogenic fungus as an endophytic to supplement IPM programs could be beneficial.

Longevity Estimates of Canary Palms and Dragon Trees via Radiocarbon Dating: Initial Results.

Correctly estimating the maximum lifespan of plant species is a necessary component of demographic and life-history studies, which, in turn, are needed for understanding climatic impacts. Arboreal monocotyledons, which can grow to >30 m in height and >5 m in trunk perimeter, are difficult to age because they do not undergo seasonal dormancy; hence, their longevity has been estimated using various size-related methods. In this study, we tested radiocarbon (14C) dating with Accelerator Mass Spectrometry (AMS) as an additional tool for determining the age of two iconic monocotyledons: the Canary Island palm and the dragon tree. A total of 25 samples were collected from the basal stem of four palms and five dragon trees on Gran Canaria and Tenerife and then processed using the most advanced 14C-AMS analysis available. Calibration curves provided by the "IntCal group" were used to determine the oldest possible age of each sample, and 16 of them were found to be "modern", i.e., formed after the 1950s. Nine samples that were either collected from exterior, but lignified, palm tissues or from interior, and lignified, dragon tree tissues suggested ages > 300 years. Given the constant improvement of 14C-AMS tools, they can contribute to the further refinement of existing scientific knowledge on Macaronesian charismatic megaflora.

Evaluation of microbial biocontrol agents for Ganoderma boninense management in oil palm nurseries

Importance of the workMicroorganisms play a pivotal role in the growth and development of plants, making their interaction a promising avenue for managing plant diseases, including those affecting oil palm cultivation. ObjectivesIn this study, endophytic microbes such as actinomycetes, bacteria, and fungi were isolated from healthy oil palm tissue in susceptible areas to serve as stock isolates. These isolates underwent in vitro evaluation to determine their efficacy in controlling Ganoderma boninense and to identify bioactive metabolites. Additionally, in planta tests were conducted using bioformulations. Materials & MethodsA dual culture assay was utilized to assess the inhibitory effects of three different stock isolates on G. boninense. To detect bioactive metabolites responsible for the inhibition, particularly phenazine and syringic acid, a series of procedures involving fermentation, extraction, and LC-MS analysis were employed. Furthermore, the efficacy of bioformulations employing various carriers and consisting of either microbial consortia was meticulously evaluated in a nursery setting. The main focus of this evaluation was to determine their capacity to effectively curb disease onset and maintain the vegetative growth of oil palm during the progression of G. boninense. ResultsSeveral strains of indigenous bacteria (Pseudomonas aeruginosa strain JQ-41, Serratia marcescens strain S16, Stenotrophomonas rhizophila strain CASMBAUDAL2), an actinobacterium (Streptomyces sp.), and a fungus (Trichoderma sp.) exhibited significant growth inhibition against G. boninense. Based on the results obtained from the LC-MS analysis of the ethyl acetate extract, it was revealed that the extract contains 2 compounds known as antifungal agents, namely phenazine and syringic acid. The most effective approach to mitigate infection involved the preventive application of bioformulations containing mixed bacterial isolates or microbial consortia. After 12 months of observation, these bioformulations demonstrated efficacy comparable to that of Marfu-P, a commercially available biofungicide for oil palm seedlings. Main findingThe study establishes the effectiveness of endophytic bioformulations as a viable strategy to manage the onset of G. boninense disease, highlighting the potential of microorganisms as an alternative solution for controlling basal stem rot in the nursery.

A functional decision support system for management of Graphiola leaf spot (Graphiola phoenicis) disease during hardening of date palm tissue cultured plants in a greenhouse

A functional decision support system (DSS) was developed through optimising values of parameters favour faster growth and development of tissue cultured plantlets of date palm and suppression of disease infection. The programming of control devices was further managed based on the ambient environmental conditions, which required variable simulation periods and a plan for controlling of all systems for maintaining the greenhouse environment. During the process of plant hardening, the plants were infected by Graphiola leaf spot (Graphiola phoenicis). The moderate temperature, and high humidity inside the greenhouse, accompanied by short days and frequent occurrence of fog during winter months, favoured the development of smut infection. It is required to manage an extended photoperiod for 16 hours with 15000 lux light intensity provided by white fluorescent tube lights and incandescent bulbs inside the greenhouse. The integrated practices of enhanced photoperiod and light intensity under the greenhouse in combination with fungicides application to plants were found very effective in suppressing the intensity of the disease and its adverse effects on the plants. Using this decision support system (DSS), the tissue cultured date palm plants cv. Barhee has been successfully hardened and managed disease and transplanted in the field for further studying establishment, survival, plant growth and fruiting-related parameters.

Functional characterization of the MSP-C6 promoter as a potential tool for mesocarp-preferential expression of transgenes

Modification of lipid composition in the mesocarp tissue of oil palm involves genetic manipulation of multiple genes. More than one mesocarp-preferential promoter is necessary for the expression of individual transgenes in the same plant to obviate transcriptional gene silencing. This study aimed to identify genes that are preferentially expressed in the mesocarp tissue and characterize selected candidate mesocarp-preferential promoters. Ten transcripts that were preferentially expressed in the mesocarp tissue were identified from the analysis of 82 transcriptome datasets of 12 different oil palm tissues. The expression of two candidate genes, MSP-C1 and MSP-C6, was verified to be preferentially expressed in the mesocarp tissues and shown to have a low expression level in non-mesocarp tissues by reverse transcription quantitative real-time PCR (RT-qPCR). MSP-C6 promoter fragments of different lengths were transformed into tomato plants for further characterization. Both unripe and ripe fruits of transgenic tomato plants transformed with a construct harboring the MSP-C6-F1 (2014 bp) promoter were shown to have high beta-glucuronidase (GUS) activities. The findings of this study suggest the potential applications of the MSP-C6 promoter as a molecular tool for genetic engineering of novel traits in fruit crops.

In Vitro Salt Stress Responses of Thai Oil Palm’s Embryogenic Callus Variety ‘SUP-PSU1’

Evapotranspiration, which includes the evaporation of water from soils, may rise as a result of climate change and rising temperatures. The outcome is that water evaporates but salt stays in the soil, raising its saltiness. The growth and development of oil palm are also affected by salt stress, but decades of time frame are required with conventional breeding due to its reproductive biological reasons and its long-time life span. However, to facilitate stress tolerance evaluation at an early stage of plant material, the in vitro selection was considered, and the evaluation process was optimized for innovating variant selection out from calluses over abiotic stress. Thus, the objectives of this research were to study the effect of concentrations of sodium chloride (NaCl) on the growth rate, electrolyte leakage (EL) and proline content of embryogenic callus (EC) of oil palm ‘SUP-PSU1’. Was also, recovery of EC growth after treating with NaCl for further evaluation to set new variant tolerant line for breeding was established. EC was treated with different concentrations of NaCl in oil palm culture medium (OPCM) supplemented with 0.1 mg L–1 3,6-Dichloro-o-anisic acid (dicamba) and 200 mg L–1 ascorbic acid (AA) for 4 weeks. The results showed that inhibition concentration at 50 % (IC50) was 149.1 mM and NaCl at high concentrations increased EL and proline content. After transferring treated EC to solidified medium without NaCl for 4 weeks, The 200 mM condition’s EC turned yellowish-brown and couldn’t withstand higher (300 and 400 mM) NaCl concentrations. The lowest growth rate was obtained with NaCl at 300 and 400 mM, and the EC entirely turned brown. So, it was judged that NaCl concentration around 300 to 400 mM can be used as a selection agent for ‘SUP-PSU1’ new variant tolerant line in oil palm tissue culture breeding condition. HIGHLIGHTS The inhibition concentration that limits the growth of embryogenic callus of oil palm ‘SUP-PSU1’ at 50 % (IC50) was 149.1 mM NaCl at high concentration resulted in an increment of electrolyte leakage (EL) and proline content The embryogenic callus of oil palm ‘SUP-PSU1’can tolerate to NaCl up to 200 mM NaCl at concentrations of 300 and 400 mM can be used as a selection agent for ‘SUP-PSU1’ new variant tolerant line in oil palm tissue culture breeding condition GRAPHICAL ABSTRACT

Endophytic microbe in stem tissue of oil palm colonized by Ganoderma sp.

Infection with Ganoderma sp. results in pseudoschlerotium, a distinctive feature of oil palm tissue trunks. The goal of this study was to study the microbial communities in Ganoderma sp. colonized oil palm trunk tissue. Endophytic bacteria are isolated from oil palm trunk tissue using selective media such as Ashby, Pikovskaya, King’s B, and a half-NA media to isolate N2 fixing bacteria, P solubilizing bacteria, Pseudomonas sp., and endophytic microbes, respectively. To prepare the sample, two methods were tested: 1) sterilizing the surface of the sample and then directly planting the stem tissue in a medium, and 2) sterilizing the surface of the tissue, crushing the tissue with a sterilizer mortar, and centrifuge for further seeding the supernatant in the selected medium. The first procedure was found to be effective in isolating N2-fixing bacteria from the top, middle, and bottom of the stem. We also recovered P solubilizing bacterium, which was yellow in colour and only located in the centre part of the stem, whereas the Pseudomonas sp. isolate was white in colour and found throughout the stem except at the bottom in the left side of the pseudosclerotium. We were able to isolate endophytic bacteria with a white hue using a half-NA medium. Method 2 did not succeed in isolating Azotobacter sp., but it can still isolate P solubilizing, Pseudomonas sp., and endophytic bacteria in all sections of the stem. The number of bacteria successfully isolated using the culture approach was low, particularly in the pseudosclerotium area. However, the larger quantity of Pseudomonas bacteria found in infected Ganoderma sp. tissue should be investigated further to establish the role of these bacteria, particularly in plant defence against Ganoderma sp.

Reproducibility of Somatic Embryogenesis Upon Resampling of Oil Palm Ortets

Somatic embryogenesis remains to be a key bottleneck in oil palm clonal propagation and resampling of embryogenic ortets may be one approach to alleviate this problem. Resampling, is currently practised by oil palm tissue culture laboratories to increase the production of clones from elite ortets. However, reproducibility of the embryogenesis outcome from resampling exercises has not been examined in detail. Therefore, embryogenesis reproducibility upon resampling was evaluated using the concordance correlation coefficient analysis. Sampling and resampling were conducted when ortets were seven to 19 years old. Resampling interval periods of three to eight years showed moderate concordance in embryogenesis reproducibility. An alternative analysis approach through categorising the ortets based on their embryogenesis outcomes into zero, low, medium and high embryogenesis categories indicated that 54.4 per cent of ortets reproduced their embryogenesis outcome while 35.6 per cent showed a shift by one embryogenesis category upon resampling. A resampling interval of 10 years was associated with a decline in embryogenesis outcomes. Observations from this study may help tissue culture laboratories in refining the selection of ortets for clonal propagation with the possibility of improving success rates in resampling exercises and thereby reducing operational costs. Keywords: Ortet, concordance, non-embryogenic, sampling interval.

Performance Comparison of Trunk Injection Mechanisms for Ganoderma Control

A severe disease known as Ganoderma Boninense, which attacks a group of wood-decaying fungi, causes Basal Stem Rot (BSR) in oil palm trees. One of the treatments introduced to control the spread is injecting a chemical into the palm's trunk. After receiving a hexaconazole treatment for a year, a study on 2008 Ganoderma-infected palm trees in an estate revealed that 95.6% of the infected palms were still standing. The injection process facilitates several devices that were used and invented. These machines utilise a pressure-injection technique that injects a chemical through a flexible hose into a healthy palm's tissue to prevent the disease from spreading. Hence, the development of effective trunk injector mechanisms for Ganoderma treatment is discussed in this article. The first injection method utilised the Pressure Injection Apparatus (PIA), which can deliver a chemical to the target sites to control BSR. Second, a Mechanical Trunk Injector mainly facilitated chemical injection into the palm trunk using a prime mover. Next, the Tractor Mounted Trunk Injector, comprised of an injection apparatus system, was found to perform effectively and efficiently. The distribution profile of the dye eosin in the stem revealed that it covered more than 80% of the cross-sectional and longitudinal axes of the palm tree's trunk. Instead of the previous injection apparatus control method, this machine could speed up the injection time. Compared to the 20-minute-per-hole completion time of the PIA, the machine only took 3 to 5 minutes to complete an injection, which was 65% faster, and about 100 to 120 palms could be covered in a day. Labour requirements were very minimal as it is a single-operated machine. Utilising the machine would also result in a reduction in the number of workers needed.

Isolation and molecular detection of Arthrobacter species grown on the surface of date palm tissue culture media

Isolation and molecular detection of <i>Arthrobacter</i> species grown on the surface of date palm tissue culture media

Date palm tissue culture and the green Morocco plan

Date palm tissue culture and the green Morocco plan

Persistence of the entomopathogenic nematode Steinernema carpocapsae on red palm weevil-infested date palm trees in an arid environment

Summary The persistence of the entomopathogenic nematode (EPN), Steinernema carpocapsae, was evaluated following application on date palm (Phoenix dactylifera) trees infested with the red palm weevil (RPW) Rhynchophorus ferrugineus in an arid environment. Live S. carpocapsae were detected in different sections of the tree trunk, including internal parts, in the soil, and from RPW cadavers. EPN third-stage infective juveniles were extracted from the palm stem tissue and soil up to 45 days and 180 days after application, respectively. Results support the high potential of commercial S. carpocapsae application as a useful biological control agent for R. ferrugineus in date palms in arid environments.

Acceleration of Callus Formation in Date Palm (Phoenix dactylifera L.) Zygotic Embryos

This study was carried out to test the effect of water soaking ofseeds of date palm ( P. dactylifera L.) as a pre-empryo excision treatment,on in vitro culture of zygotic empryos. The objective was optimization oforganogenic callus formation. Mature seeds of “Barakawi” and “MishrigWad Laggi” date palm cultivars were used as sources of embryos.Durations of 0- up to 9-days of seed water soaking, prior to embryoexcision and culture, were tested. Murashige and Skoog (1962) mediumwas used as basal medium. The cultures responded positively to the watersoaking treatments and resulted in the organogenic responses of swelling,germination, callus and adventitious roots formation were obtained. Themagnitude and type of response varied with cultivar and soaking duration.All water soaking treatments increased the measured responses relative tothe control. The results showed that a 6-day soaking treatment wasoptimal for all measured responses compared with the other periods ofsoaking tested. Calli formation in 6-day water soaking treatment were50% and 70% for “Barakawi” and “Mishrig Wad Laggai” ,respectively.However, increasing the soaking duration beyond the 6-day had a markednegative effect on morphogenic response regardless of cultivar. The bestresults for all measured parameters were obtained with embryos of“Mishrig Wad Laggi” compared with embryos excised from “Barakawi”.It seems that soaking date palm seeds in water prior to embryo excisionplay an important role, in enhancing morphogenic response and callusformation in date palm tissue culture.

A Microrobot With an Attached Microforce Sensor for Transurethral Access to the Bladder Interior Wall

AbstractWe present the conceptual design and limited functionality prototype and characterization of a system for application in transurethral palpation of any targeted area of the bladder interior wall tissue consisting of a robotic manipulator and a microforce sensor attached at its tip all less than 3.5 mm in diameter. A hyper-redundant ten-joint six degrees-of-freedom (6DOF) manipulator (5DOF rigid and five-joint continuum segments) is presented along with the forward and inverse kinematics analyses based on a Jacobian formulation to prevent configuration singularities. Simulated motion studies demonstrate the ability of the proposed manipulator to attain a desired pose (normal to the tissue) with any area in the bladder including the difficult to reach trigone area. A strain gauge-based microforce sensor is designed using finite element analysis (safety factor &amp;gt; 3), prototyped using additive manufacturing, and characterized. The characterized sensor was used to acquire in vivo measurements to evaluate human palm tissue viscoelastic properties. A single module of the continuum segment is designed and prototyped using additive manufacturing, and used to characterize its tension-bend angle behavior. Finite element analysis is used to improve structurally weak regions of the vertebra. A three-joint four-vertebrae prototype was successfully actuated to reach a bend state using tendons. The developed robot and sensor prototypes demonstrate capabilities of the proposed concept which could be a possible solution to quantitatively evaluate localized biomechanical properties of the bladder tissue to improve treatment and provide better patient care.

Genome-Wide Identification of Homeodomain Leucine Zipper (HD-ZIP) Transcription Factor, Expression Analysis, and Protein Interaction of HD-ZIP IV in Oil Palm Somatic Embryogenesis

Understanding the molecular mechanisms underlying somatic embryogenesis is essential for resolving the problems related to the long duration of the process and a low rate of somatic embryo induction in oil palm tissue culture. In this study, we conducted genome-wide identification of the oil palm homeodomain leucine zipper (EgHD-ZIP) family, which is one of the plant-specific transcription factors reported to be involved in embryogenesis. EgHD-ZIP proteins can be divided into four subfamilies, which have similarities in gene structure and protein-conserved motifs within a group. In silico expression analysis showed that the expression of EgHD-ZIP gene members in the EgHD-ZIP I and II families, as well as most members in the EgHD-ZIP IV family, were up-regulated during the zygotic and somatic embryo developmental stages. In contrast, the expression of EgHD-ZIP gene members in the EgHD-ZIP III family was down-regulated during zygotic embryo development. Moreover, the expression of EgHD-ZIP IV genes was validated in the oil palm callus and at the somatic embryo stages (globular, torpedo, and cotyledon). The results revealed that EgHD-ZIP IV genes were up-regulated at the late stages of somatic embryogenesis (torpedo and cotyledon). While BABY BOOM (BBM) gene was up-regulated at the early stage of somatic embryogenesis (globular). In addition, the Yeast-two hybrid assay revealed the direct binding between all members of the oil palm HD-ZIP IV subfamily (EgROC2, EgROC3, EgROC5, EgROC8, and EgBBM). Our findings suggested that the EgHD-ZIP IV subfamily and EgBBM work together to regulate somatic embryogenesis in oil palms. This process is important because it is widely used in plant biotechnology to produce large quantities of genetically identical plants, which can be used for oil palm tissue culture improvement.