Somatic Embryo Formation Research Articles

Optimization of crop tissue culture technology and its impact on biomolecular characteristics

Modern agriculture relies on crop tissue culture technology for fast propagation, genetic improvement, and protection of plant species. Conventional media like Murashige and Skoog (MS) usually lack optimal conditions for embryogenesis, necessitating the development of improved media tailored to specific crop requirements. In this article, we introduce an Efficient Grid Identified-Deep Feedforward Neurons (EGI-DFFN) to identify the ideal nutrient and vitamin levels of the crop plants for improving crop tissue culture aimed to improve crop plant growth in a lab setting by predicting callogenesis rate (CGR), embryogenesis rate (EGR), and somatic embryo number (SEN), shoot regeneration rate (SRR), rooting rate (RR).Different concentrations of ionic macronutrients, bio-molecular, and vitamins of the crop plant are the input to the predictive model, which is collected through the laboratory Callus Induction Experiment (CIE). Z-score normalization is used to preprocessing the CIE data to ensure consistent scales across different input features and improve model training performance. DFFN used discriminates to predict complex relationships and interactions between CGR, EGR, SEN, SRR, and RR with EGI tuning. The EGI-DFFN model has significantly improved crop tissue culture growth by accurately predicting the CGR, EGR, SEN, SRR, and RR respectively. The EGI-DFFN model enhances understanding of how ionic macronutrients and vitamins impact plant growth. It identifies optimal concentrations of the biomolecular to enhance somatic embryo formation and plantlet development, providing insights for optimizing crop tissue culture conditions for optimal growth outcomes.

Autophagy is essential for somatic embryogenesis in citrus through regulating amyloplast degradation and lipid homeostasis.

Autophagy is a conserved degradation pathway that regulates the clearance of paternal substrate at the early embryogenesis stage of animals. However, its mode of action is likely different in plants, which can regenerate through apomixis without fertilisation. Somatic embryogenesis (SE) is a unique plant process widely used for plant propagation and germplasm utilisation. Here, we studied citrus as an example and found a higher autophagic activity after SE initiation. Interestingly, amyloplasts were frequently found inside autophagosomes, whereas the inhibition of autophagy blocks amyloplasts/starch degradation and hinders somatic embryo formation. Furthermore, the consumption of storage lipids was faster in autophagy mutants, suggesting lipid metabolism is activated when starch utilisation is blocked. Exogenous application of autophagy-inducing chemicals (e.g. spermidine) significantly promoted the formation of autophagosomes and increased SE efficiency, indicating a positive correlation between autophagy, energy metabolism, and somatic embryo formation in citrus. Taken together, our study unveils a pathway for the degradation of plant-specific organelles and provides an effective approach for plant propagation.

High Efficiency of the Somatic Embryogenesis Sequences of Desert Lime (Eremocitrus Glauca)

Abstract: Eremocitrus glauca is a desert plant that is sources to the plant genetic resources for citrus, because these plants are resistant cultivars to some enemy mainly in the tropical region. Isolated protoplast culture media supplemented with to 2-isopentenyladenine (2iP) maintaining under the light intensity range 13.23. mol/m2/s1 to 70.8 mol/m2/s1 sigificantly effect to plating effisiensy protoplasts, globular somatic embryos, and plant regeneration of Eremocitrus glauca. The temperature emission and light illumination are applicate to cells has implicated to cell recovery and formation of somatic embryos. 2iP and environment factors increased the health cell from enzyms stress, grow and developed rapidly. In vitro temperature for somatic embryo formation in cultures is achieved on 25oC in corporations with 0.1 mg/l, 2 iP and more likely in pattern linear, with equation Y = 0,7336x + 4,1894, R2 = 0.8172. The same patern is also accur to ligh illumination. Attractive conclusion that light and temperature must be well to do on plant physiology and chemistry. The sequence from isolated protoplast to cell recovery, formation of somatic embryos and plant or shoot regeneration under control of media, plant growht regulators and environment factors has establish in Eremocitrus glauca. Forturmore the gene tranfer from this species has been easily.

Somatic embryogenesis and plant regeneration from leaf callus with genetic stability validation using SCoT markers in Paramignya trimera, a medicinal plant native to Vietnam

Xao tam phan (Paramignya trimera (Oliv.) Guillaum) is a medicinal plant native to Vietnam, that is renowned for its therapeutic properties, particularly for the treatment of various ailments, including cancer. This study investigated in vitro propagation of P. trimera through somatic embryogenesis and shoot organogenesis using leaf callus. Various culture media, plant growth regulators, malt extract, and carbon sources were evaluated to optimize callus induction and somatic embryo formation from leaf explants. DNA barcoding confirmed 96.96% to 100% homology with P. trimera specimens from Khanh Hoa province, Vietnam. The highest callus formation rate, reaching 100%, was observed in one-year-old explants cultured in Woody Plant Medium (WPM) supplemented with 2.0 mg L-1 naphthaleneacetic acid (NAA) and 0.2 mg L-1 benzylaminopurine (BAP) in the dark for over six weeks. In WPM supplemented with 30 g L-1 sucrose, 4.0 mg L-1 BAP, and 500 mg L-1 malt extract, globular stage embryos developed into embryoids and shoots and buds clumped at 10 and 18 weeks, respectively. Shoot organogenesis was observed in WPM supplemented with 30 g L-1 sucrose and 0.07 mg L-1 thidiazuron (TDZ) after 18 weeks of culture. Genetic fidelity assessments using 12 SCoT markers indicated that in vitro plantlets were homologous to the mother plant. This study provides a viable method for the conservation and sustainable cultivation of Xao tam phan, ensuring a stable supply of this valuable medicinal resource.

Morpho-histology of co-occurrence of somatic embryos, shoots, and inflorescences within a callus of Limonium 'Misty Blue'.

This is the first attempt to report the co-occurrence of somatic embryos, shoots, and inflorescences and their sequential development from stem cell niches of an individual callus mass through morpho-histological study of any angiosperm. In the presence of a proper auxin/cytokinin combination, precambial stem cells from the middle layer of a compact callus, which was derived from the thin cell layer of the inflorescence rachis of Limonium, expressed the highest level of totipotency and pluripotency and simultaneously developed somatic embryos, shoots, and inflorescences. This study also proposed the concept of programmed cell death during bipolar somatic embryo and unipolar shoot bud pattern formation. The unique feature of this research was the stepwise histological description of in vitro racemose inflorescence development. Remarkably, during the initiation of inflorescence development, either a unipolar structure with open vascular elements or an independent bipolar structure with closed vascular elements were observed. The protocol predicted the production of 6.6 ± 0.24 and 7.4 ± 0.24 somatic embryos and shoots, respectively, from 400 mg of callus, which again multiplied, rooted, and acclimatised. The plants' ploidy level and genetic fidelity were assessed randomly before acclimatisation by flow cytometry and inter simple sequence repeats (ISSR) marker analysis. Finally, the survivability and flower quality of the regenerated plants were evaluated in the field.

A WRI1-dependent module is essential for the accumulation of auxin and lipid in somatic embryogenesis of Arabidopsis thaliana.

The potential for totipotency exists in all plant cells; however, the underlying mechanisms remain largely unknown. Earlier findings have revealed that the overexpression of LEAFY COTYLEDON 2 (LEC2) can directly trigger the formation of somatic embryos on the cotyledons of Arabidopsis. Furthermore, cotyledon cells that overexpress LEC2 accumulate significant lipid reserves typically found in seeds. The precise mechanisms and functions governing lipid accumulation in this process remain unexplored. In this study, we demonstrate that WRINKLED1 (WRI1), the key regulator of lipid biosynthesis, is essential for somatic embryo formation, suggesting that WRI1-mediated lipid biosynthesis plays a crucial role in the transition from vegetative to embryonic development. Our findings indicate a direct interaction between WRI1 and LEC2, which enhances the enrichment of LEC2 at downstream target genes and stimulates their induction. Besides, our data suggest that WRI1 forms a complex with LEC1, LEC2, and FUSCA3 (FUS3) to facilitate the accumulation of auxin and lipid for the somatic embryo induction, through strengthening the activation of YUCCA4 (YUC4) and OLEOSIN3 (OLE3) genes. Our results uncover a regulatory module controlled by WRI1, crucial for somatic embryogenesis. These findings provide valuable insights into our understanding of plant cell totipotency.

Transcriptional Profiling Reveals Key Regulatory Roles of the WUSCHEL-Related Homeobox Gene Family in Yellowhorn (Xanthoceras sorbifolia Bunge)

The WUSCHEL-related homeobox (WOX) gene family plays a crucial role in regulating embryonic development, organ formation, and stress resistance. Yellowhorn (Xanthoceras sorbifolia Bunge), a drought-resistant tree known for its oil production, lacks sufficient information regarding the WOX gene family. To understand the evolutionary mechanisms and potential functions of this gene family in yellowhorn, we conducted a comprehensive investigation on its expression patterns and evolutionary characteristics. Our analysis revealed the presence of nine XsWOX genes in the yellowhorn genome, which could be categorized into three distinct clades through a phylogenetic analysis. A chromosomal localization analysis indicated that these nine XsWOX genes were situated on six out of the fifteen chromosomes. An intra-species collinear analysis revealed only one pair of tandem duplicated genes within the XsWOX family. The promoter regions of the XsWOX family were found to contain responsive cis-acting elements associated with plant growth and development, stress responses, and hormone signaling. Moreover, an analysis of the gene expression profiles in different developmental stages of callus revealed significant expressions of XsWOX1, XsWOX4, and XsWOX5 in embryogenic callus and somatic embryo formation, suggesting that they have special roles in regulating yellowhorn’s somatic embryogenesis. Furthermore, the expression level of XsWOX5 indicated its potential involvement not only in organ formation but also in responding to low temperature, salt, and saline-alkali stresses. Overall, our findings lay a solid foundation for future in-depth studies on the functionality and evolution of XsWOX genes in yellowhorn.

H2O2 Significantly Affects Larix kaempferi × Larix olgensis Somatic Embryogenesis.

Larch is widely distributed throughout the world and is an important species for timber supply and the extraction of industrial raw materials. In recent years, the hybrid breeding of Larix kaempferi and Larix olgensis has shown obvious heterosis in quick-growth, stress resistance and wood properties. However, its growth and development cycle is too long to meet general production needs. In order to shorten the breeding cycle, we have for the first time successfully established and optimized a somatic embryogenesis system for Larix kaempferi × Larix olgensis. We found that the highest rate of embryonal-suspensor mass (ESM) induction was observed when late cotyledonary embryos were used as explants. The induced ESMs were subjected to stable proliferation, after which abscisic acid (ABA) and polyethylene glycol (PEG) were added to successfully induce somatic embryos. Treatment with PEG and ABA was of great importance to somatic embryo formation and complemented each other's effect. ABA assisted embryo growth, whereas PEG facilitated the formation of proembryo-like structures. On top of this, we studied in more detail the relationship between redox homeostasis and the efficiency of somatic embryogenesis (frequency of ESM induction). During subculture, we observed the gradual formation of three distinct types of ESM. The Type I ESM is readily able to form somatic embryos. In contrast to type I, the type III ESM suffers from severe browning, contains a higher level of hydrogen peroxide (H2O2) and demonstrates a decreased ability to form somatic embryos. External treatment with H2O2 decreased the somatic embryogenesis efficiency of Type I and type III ESMs, or the higher the exogenous H2O2 content, the lower the resulting somatic embryogenesis efficiency. We found that treatment with the H2O2 scavenger DMTU (dimethylthiourea) could significantly increase the somatic embryogenesis efficiency of the type III ESM, as a result of a decline in endogenous H2O2 content. Overall, these findings have contributed to setting up a successful somatic embryogenesis system for larch production.

Somatic Embryogenesis and Direct Shoot Bud Formation from in vitro Root Segments of Garlic (Allium sativum L.)

In vitro techniques are unconventionally used for garlic improvement. In garlic tissue culture, numerous roots are produced in vitro. Segments (1 cm) of those in vitro roots were cultivated on MS medium with 5.0 μM 2, 4-D for callus initiation followed by transfer to 0.5 μM 2, 4-D for somatic embryogenesis. Root segments had 51.67% callus and somatic embryo formation. Plantlet regeneration was obtained from 90% calli after being transferred to MS medium supplemented with 5.0 μM kinetin. Within 6 months, 54.8 plants per root segment and 42.2 plants per root tip explant were found. MS media supplemented with 1.0 or 5.0 μM NAA in combination with 10.0 or 50.0 μM BA were used for direct shoot bud induction from root segments. The highest percentage (23.33 %) of direct shoot bud regeneration was found in 5.0 μM NAA with 50.0 μM BA within 2 months. The number of shoots per explant was varied from 1-81 with the highest average of 15.75 shoots per explant. The shoots produced bulblets upon transfer to a medium containing higher amount of sucrose (12%). The largest bulblets weighed 356.57 g in the medium with 5.0 μM NAA and 50.0 μM BA. In both pathways, regeneration occurred from only the apical (distal) end of the root segments. The protocols are promising for continuous regeneration of propagules and genetic transformation study for improvement of garlic. Plant Tissue Cult. & Biotech. 33(2): 135-142, 2023 (December)

Cloning, Characterization, and Expression Pattern Analysis of the BBM Gene in Tree Peony (Paeonia ostii)

BABY BOOM (BBM) is one of the members of the plant-specific APETALA2/ethylene-responsive factor (AP2/ERF) transcription factor superfamily. It acts as a key regulator of plant cell pluripotency, playing a significant role in promoting somatic embryogenesis. In this study, a BBM gene named PoBBM was screened, cloned, and identified from the third-generation full-length transcriptome data of Paeonia ostii. Its open reading frame was 2136 bp, encoding 711 amino acids. Sequence feature analysis revealed that it possessed two AP2 conserved domains and eight motifs, including bbm-1. The phylogenetic tree indicated that PoBBM clusters with AtBBM in the euANT group of the Arabidopsis AP2 family, which is most closely related to grape VvBBM and may have the same ancestry as grape. Subcellular localization demonstrated that the PoBBM protein was localized in the nucleus. Semi-quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to assess the PoBBM transcript levels during ten developmental stages of somatic embryos and in five tissue types of peonies. The results indicate that PoBBM was highly expressed in the early stages of peony somatic embryo development. The expression on 0–15 d was the highest and decreased gradually with somatic embryogenesis. The gene is almost not expressed after 40 d since somatic embryo formation. PoBBM was expressed in roots, stems, leaves, seeds, and calli, with the highest levels in seeds, followed by leaves and calli. The PoBBM protein displayed transcriptional self-activation activity, which may facilitate further research on its relationships with other proteins. The above results provide a key gene PoBBM for somatic embryogenesis in peonies, which is significant for advancing the establishment of a stable and efficient regeneration and genetic transformation system for peonies.

Development of a system for the formation of transgenic somatic embryos in the liquid medium in Medicago truncatula

Somatic embryogenesis is the formation of embryos from plant’s somatic cells. It is widely used in biotechnology for reproduction of plants, studying of regeneration process and it also represents a convenient way to obtain transgenic plants. Currently, a solid medium is usually used for the formation of transgenic somatic embryos, which has a number of disadvantages.
 We are developing a system for cultivating explants in a liquid medium for the transformation and formation of somatic embryos forMedicago truncatula. Unlike a solid medium, it should allow using petioles as explants, simplify the renewal of the medium, replace disposable cultivation containers with reusable ones, and also reduce the time required for the formation of somatic embryos.
 Currently, the optimal concentration of hygromycin as a selective agent in such a system was found. Interestingly, it appeared to be lower than the selective hygromycin concentration in a solid medium.
 The addition of cefotaxime to the medium reduces the number of somatic embryos formed, but does not completely suppress their formation. Thus, cefotaxime can be used to eliminate agrobacteria during transformation using this cultivation system.
 Embryos withGUSoverexpression transformed with this method were successfully obtained.

The screening vector system of morphogenic regulators in Fabaceae

Legumes are important agricultural and food crops, however, some legume species have a low regenerative capacityin vitro, which complicates obtaining the genetically modified plants with improved properties and analysing gene function.
 To search for genes that stimulate somatic embryogenesis and to increase the regeneration frequency in legumesin vitro, we designed a screening vector system that will allow faster cloning of genes encoding potential regulators of morphogenesis by preserving restriction sites in the final vector. The construction of vectors is based on the Golden Gate [1] modular cloning method. Using type II restriction endonucleases, DNA fragments form sticky ends and are combined in a given order to form multigene constructs intended forAgrobacterium-mediated transformation. In order to identify efficient variants for gene expression, we used a number of promoters: CaMV 35S(long), CaMV 35S(double), nopaline synthase (nos), actin 2 (act2), and a number of terminators: 35S CaMV, nos, act2 forMtWOX9-1overexpression.Medicago truncatulaWOX9-1 (MtWOX9-1) is a WUSCHEL- related homeobox transcription factor for which a positive effect on the formation of somatic embryos in callus culture was previously shown [2].
 Based on the analysis of embryogenic tissue, the optimal combination of promoters and terminators will be selected to assemble the vector for screening of morphogenetic regulators.
 The work is supported by the Ministry of Science and Higher Education of the Russian Federation (Agreement 075-10-2021-093, Project PBB-RND-2243).

The effect of H<sub>3</sub>BO<sub>3</sub> and 2,4-D on <i>in vitro</i> Callus and Somatic Embryo Formation of Bitter Melon (<i>Momordica charantia</i> L.)

The effect of H<sub>3</sub>BO<sub>3</sub> and 2,4-D on <i>in vitro</i> Callus and Somatic Embryo Formation of Bitter Melon (<i>Momordica charantia</i> L.)

Production of Black Cumin via Somatic Embryogenesis, Chemical Profile of Active Compounds in Callus Cultures and Somatic Embryos at Different Auxin Supplementations

Black cumin or Nigella sativa L. is a medicinal plant of the Ranunculaceae family that has enormous importance. It has traditionally been used to cure a lot of diseases since ancient times. In the current study, the effects of different auxins on callus induction and subsequent somatic embryo formation of N. sativa L. cv. Black Diamond were examined. The best result of callus induction was observed when cotyledon explants were incubated in a Murashige and Skoog (MS) medium supplemented with 1.0 mg L−1 α-naphthaleneacetic acid (NAA). The formation of somatic embryos was achieved efficiently from cotyledon-derived calli cultured on a 2 mg L−1 Indole-3-butyric acid (IBA)-containing medium. Furthermore, histological analysis of embryogenic calli was used to detect the presence of different developmental stages of somatic embryos. In contrast to the calli and embryos of N. sativa ‘Black Diamond’, which initiated in the dark, light was necessary for the complete differentiation of callus and embryo cultures into shoots/developed plants. Hypocotyl-derived calli and embryos were successfully differentiated on IBA at 2.0, 1.0 mg L−1, and NAA at 2.0 mg L−1. To the best of our knowledge, this work can be considered the first report on the differentiation of N. sativa ‘Black Diamond’ somatic embryos into developed plants. Moreover, the metabolic profiles of secondary products of N. sativa ‘Black Diamond’ callus and embryo cultures originated from the best auxin treatments identified and were compared with that of intact seeds. Callus cultures of N. sativa ‘Black Diamond’ contained thymoquinone (TQ) in a significant percentage of the peak area (2.76%). Therefore, callus cultures could be used as a perfect alternative source of TQ for pharmaceutical and therapeutic purposes. In addition, fatty acids and/or their esters were recorded as the major components in callus and embryo cultures. These vital compounds could be isolated and used for numerous industrial applications.

Somatic embryo formation, plantlet vigorous and greenhouse preliminary test of garlic bulblet obtained through somatic embryogenesis

Garlic production in Indonesia is still low due to limited availability of seedling. Approximately 10% of garlic yield are used for the next cultivation. Therefore about 90 % of garlic consumption are still imported from China. In addition to conventional seed production, alternative routes are needed, such as through somatic embryogenesis. Research consists of three activities, namely 1) induction and multiplication of callus, 2) embryo somatic and plantlet formation and 3) preliminary test of bulblet in vitro in the greenhouse. Parameters observed in the laboratory activities were time of callus emergence, callus weight and morphology, time of somatic embryo emergence, embryo diameter and weight per bottle, plantlet number and vigorous and bulblet number per bottle, while in the greenhouse, parameter observed were survival of bulblet number, plant height, leaf number per plant, and clove number and weight. The result showed that application of 2,4-D 3 mg/l with casein hydrolysate acid was suitable for callus induction of garlic var. Lumbu Kuning, while BAP 2 mg/l was the most suitable formula for callus multiplication, the addition of paclobutrazol 1 ppm and sucrose 20% in MS medium was the most suitable treatment for plantlet vigorous, while the bulblet formation could be developed by using jasmonic acid with sucrose of 12%. The best medium for growjng media of in vitro bulblet in the greenhouse was the combination of sandy soil, cocopeat and manure.

Collectible Cell Lines of Larix sibirica Obtained by Somatic Embryogenesis and Their Ability to Regenerate

A protocol for the cultivation of Larix sibirica Ledeb. by somatic embryogenesis was developed (RF Patent No. 2456344, 2012). The L. sibirica collection consisted of 22 actively proliferating cell lines (CLs) obtained from immature zygotic embryos. The age of CLs ranged from 1 to 14 years. CLs differed in their growth intensity, embryonic productivity, hormonal balance, and genetic stability, as well as in their regenerative ability. In most proliferating CLs, the formation of globular somatic embryos continued for 2–4 years. Here, a number of CLs actively proliferated for 9–14 years or more. The formation of embryogenic cultures in L. sibirica is associated with the content of phytohormones and their localization in embryo cells. The cytogenetic studies revealed the genetic stability of young CLs (up to 1 year), in which the karyotype consisted of the diploid number of chromosomes (2n = 24). Genomic mutations were observed in the long-term proliferation of CLs. Individual CLs can maintain cytogenetic stability for many years; such CLs can successfully be used to preserve germplasm, obtain planting material, and for plantation reforestation.

The Effects of Different Auxin–Cytokinin Combinations on Morphogenesis of Fritillaria meleagris Using Bulb Scale Sections In Vitro

Fritillaria meleagris is a horticulturally and medicinally valuable bulbous plant that requires a period of low temperatures for proper growth and flowering. Since conventional methods of propagation are ineffective and very slow, tissue culture techniques offer an integrated approach to mass production of this valuable geophyte. In this study, we investigated the effects of various auxin–cytokinin combinations on different morphogenetic pathways in bulb scale culture. Bulbs obtained in vitro were cut longitudinally, and bulb scales were cultured for four weeks at 7 °C on MS medium supplemented with 6-benzylaminopurine (BAP) in combination with 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthaleneacetic acid (NAA) at different concentrations in order to investigate the influence of plant growth regulators (PGRs) on different morphogenetic responses. Regeneration percentage, number of shoots per explant, shoot length, number of bulbs and number of somatic embryos were monitored weekly. After chilling, bulb scales were transferred to 24 °C, and all parameters were recorded again. Low PGR concentrations were very effective for shoot multiplication, yielding up to 5.5 shoots per explant. 2,4-D (at 2 mg/L) in combination with low BAP (0.25 mg/L) produced the highest number of bulbs (11.00 ± 0.00), while PGR-free medium was extremely effective in somatic embryo formation (13.50 ± 2.90). Detached somatic embryos and bulblets continued to grow and develop on fresh PGR-free medium. We present data demonstrating that low auxin–cytokinin concentrations and PGR-free medium provide an effective method for a combined morphogenetic pathway in F. meleagris that is suitable for large-scale propagation.

Transcriptome and physiological analysis highlights the hormone, phenylpropanoid, and photosynthesis effects on early somatic embryogenesis in Ginkgo biloba

The somatic embryogenesis (SEs) system in Ginkgo biloba L. has not been fully established due to the recalcitrance of this species, and it has seriously limited the application of SEs technology, such as elite varieties large-scale production, gene editing, or transformation. Recently, we have induced the cotyledon-shaped somatic embryos (SE) of G. biloba, which was so far observed the closest to mature somatic embryos. Here, we performed the physiological analysis in six cultured stages during SEs formation, including d-NEC (non-embryogenic callus), d-EC (embryogenic callus), d-SE, d-WHC (while hairy callus), L-EC (EC in light), and L-SE (SE in light). The transcriptomic analysis in d-NEC, d-EC, L-EC, and d-WHC was also carried out. We found that the higher (IAA+ZR+iPA)/ABA and the lower IAA/(iPA+ZR) levels were critical in the somatic embryogenesis of G. biloba. The higher levels of soluble sugar, total flavonoid, terpene lactone, PAL, F3'H, and CCR were beneficial to somatic embryogenesis. In addition, the peroxidase (POD) might be related to the cell wall formation of somatic embryos in G. biloba. A total of 36517 unigenes were identified in four groups by transcriptome analysis, and genes differentially expressed (DEGs) were related to hormone homeostasis i.e., SAUR50, Jasmonic acid synthesis i.e., OPR3, phenylpropanoid biosynthesis i.e., CCR1, POD43 and C4H, terpenoid backbone biosynthesis i.e., HMGS, starch and sucrose metabolism, i.e., coniferin β-glucosidase, and photosynthesis, i.e., D1 protein. Furthermore, the majority of identified up-regulated transcription factors (TFs) such as MYB4, WRKY43, EFR8, and WUS might be responsible for the acquisition of SEs. The light was helpful to the early formation of somatic embryos, and the gene GbWUS1 and GbCLV3 were detected with higher expression in early SE induction of G. biloba. This finding will provide important theoretical and technical support for gene editing, cell totipotency research, and rapid propagation in G. biloba.

Detection of <i>AtRKD4</i> Gene and Induction of Somatic Embryo in Transformant of <i>Phalaenopsis amabilis</i> Carrying 35S::GR::<i>AtRKD4</i>

Phalaenopsis amabilis (L.) Blume is a native Indonesian orchid that plays an important role in the breeding of orchid’s hybrid worldwide. The high consumer demand causes a decline in the population of orchids for commercial trade. Plant propagation through induction of somatic embryogenesis will be very beneficial, because plants can be obtained in large numbers and uniform. AtRKD4 gene is an important gene in the model plant Arabidopsis thaliana which functions very early in development stage to initiate embryo formation. The AtRKD4 gene has been inserted into the P. amabilis orchid and several transformants have been obtained. This study aims to determine the stability integration of the AtRKD4 gene in the transformant P. amabilis genome and induce somatic embryo formation on transformant orchids. Plantation of leaf explants from P. amabilis transformants on hormone-free New Phalaenopsis basic medium induced somatic embryo formation by 20%. Anatomical analysis showed that there is no difference stage between anatomy of somatic embryo development pattern in P. amabilis transformant and somatic embryo development pattern of monocot plants in general, PCR analysis using AtRKD4 specific primers showed that the embryos still contained 198 bp fragments of the AtRKD4 gene. In conclusion, the AtRKD4 gene is stably integrated in the P. amabilis orchid genome and can continuously induce the formation of somatic embryo from somatic cells of orchid transformants.

Colchicine-sensitivity Test in Cassava Leaf Lobes and its Effect on Callus and Somatic Embryo Formation

Background: Colchicine acts as a polyploidy inducer but at high concentrations, it causes high cell mortality. To improve the efficiency of colchicine polyploidization in cassava, leaf lobes colchicine-sensitivity tests were carried out and LD50 determined at 0.00, 0.05, 0.10, 0.20 and 0.25g/l colchicine concentrations in the varieties Ankrah, Dagati, Tomfa and Tuaka. Methods: Colchicine treated leaf lobes were regenerated into callus on either 8 mg/l 2, 4-D or Picloram. The calli were subsequently regenerated into somatic embryos by NAA. LD50 of 0.09, 0.11, 0.13 and 0.09 mg/L colchicine concentration were determined for Ankrah, Dagati, Tomfa and Tuaka respectively in 2, 4-D. Similarly, LD50 of 0.12, 0.10, 0.14 and 0.10 mg/L were respectively obtained in Picloram. Result: In 2, 4-D, Ankrah and Tuaka were more sensitive to colchicine than Dagati and Tomfa whereas in picloram, Dagati and Tuaka showed more sensitivity. Callus proliferation differed significantly among varieties and influenced by the concentration of colchicine.