In vitro induced cold memory fails to enhance embryogenic cultures development in Araucaria angustifolia Abstract
This research aimed to study how exposing somatic embryos of Araucaria angustifolia to cold temperatures in vitro could enhance somatic embryogenesis. The somatic embryos were induced from immature zygotic embryos, grown for 120 days, and then subjected to 4.5°C for 0, 2, 4, 8, or 16 days. After the cold treatment, the embryogenic cultures were returned to 25±2°C for 20 days and examined using light microscopy and cytochemistry. It was found that the embryogenic cultures at the PEM III stage did not show further development, and the growth of the embryogenic masses was negatively affected in the longer-duration cold treatment. Overcoming these challenges in the somatic embryogenesis protocol for A. angustifolia requires further integrative investigations on epigenetic events driving the zygotic embryo development.
Key words
Biotechnology; Brazilian pine; critically endangered species; plant cell culture
INTRODUCTION
The process of somatic embryogenesis in Araucaria angustifolia (Bertol.) O. Kuntze is a crucial biotechnological tool for the in vitro propagation of this species (Stefenon et al. 2009, 2020). This process involves inducing embryogenic cultures from the apex of immature zygotic embryos at the early stage of globular development when the highest levels of endogenous auxin are present (Astarita et al. 2003). It leads to the formation of pro-embryos, indicating the initial stages of embryogenesis. However, despite extensive research efforts, achieving complete plant development from somatic embryos remains a significant challenge for A. angustifolia, and new procedures must be explored (Stefenon et al. 2020).
The development cycle of a zygotic embryo of A. angustifolia, from the emergence of the cones to seed maturation, takes about two years (Mantovani et al. 2004, Goeten et al. 2020). The developing seeds go through two winter seasons with temperatures below 10 °C during this period. The development of the early embryo at an advanced stage occurs at the beginning of the last winter season of the cycle, shortly before seed maturation (Mantovani et al. 2004, Goeten et al. 2020). The existence of an epigenetic memory of cold has been demonstrated for overwintering crops (Niu et al. 2024), which require periods with temperatures below 10 ˚C to promote flowering. Due to the difficulty in advancing the somatic embryogenesis of A. angustifolia beyond the later pro-embryogenic masses (PEM III) stage, this study investigated the induction of a putative cold memory of somatic embryos through exposure to low temperatures for different periods, aiming to advance PEM III cultures into further stages.
MATERIALS AND METHODS
Early-stage pine cones were gathered in Curitibanos, Santa Catarina (27°17’02.7”S, 50°32’05.5”W) during the summer season, in January 2023. The cones were cleaned using a neutral detergent, rinsed with distilled water, and dried. Viable seeds were carefully chosen and disinfected in 70% ethanol for ten minutes, followed by a 2% sodium hypochlorite solution with Tween20 for 30 minutes, and triple-rinsed with autoclaved distilled water.
Embryos in the early-to-late transition phase and late embryos were extracted from the seeds and placed in Petri dishes containing BM culture medium (Gupta & Pullman 1991) solidified with 2.0 g L-1 Phytagel® and supplemented with 30 g L-1 sucrose, 5 μM 2,4-D, 2 μM kin, and 2 μM BAP (Silveira et al. 2002). The pH of the medium was adjusted to 5.8 before autoclaving at 121 °C, 1.3 atm, for 15 minutes. The zygotic embryos were kept in this culture medium at 25±2 °C in the dark for 120 days to induce somatic embryogenesis, with monthly subculturing. Following this period, five culture lineages were selected and divided into five plates for each lineage, totaling 25 plates with five embryonic cell clusters in each, setting up an experiment with five biological replicates and five technical replicates. The plates were then kept for 30 days at 25±2 °C without light to allow the embryogenic cultures to grow, using the same culture medium as in the induction phase. The developmental stage of the embryogenic cultures after this period was assessed through cytochemical analysis using acetocarmine and Evan’s blue staining (Steiner et al. 2015) and observed under a light microscope Olympus® BX-40, with images recorded using an Olympus DP71 camera.
After 30 days of growth, one plate of embryogenic masses from each lineage was cultivated in the dark for two, four, eight, or 16 days at 25±2 °C (control treatment) or 4.5 °C. Following each cold treatment, the plates were moved from the cold and cultured in the dark at 25±2 °C for 20 days. After this period, the embryogenic masses were examined under a stereomicroscope for morphological evaluation. Additionally, the cultures were double stained with acetocarmine and Evan’s blue and observed under a light microscope to assess the developmental stage of the somatic embryos.
RESULTS AND DISCUSSION
In different regions of the explants, two distinct types of calluses were observed: one with a yellowish color and limited development (Figure 1a), and another with a white translucent appearance and friable texture (Figure 1b). In some cases, late embryos germinated (Figure 1c) instead of developing into embryogenic cultures. The white translucent calluses exhibited embryogenic characteristics when stained with acetocarmine and Evan’s blue. Red-stained cell masses surrounded by blue-stained suspensor-like cells, characteristic of the PEM III stage, were visible (Figure 1d).
Induction of somatic embryogenesis in A. angustifolia. (a) Callus with yellowish coloration. (b) Embryogenic callus with friable consistency and white translucent coloration. (c) Latte germinated zygotic embryo in the induction medium. (d) Cytochemical analysis of latte pro-embryogenic mass (PEM III) under light microscopy after double staining with acetocarmine and Evan’s blue.
After subjecting the samples to cold treatment and allowing them to grow at 25±2 °C, cytochemical analysis revealed that all treatments showed embryogenic masses at the PEM III stage (Figures 2a-h). The analysis indicated that there were no visible advances in the development of the embryogenic masses compared to their developmental stage at the time of introduction to 4.5 °C. Furthermore, a morphological evaluation suggested that the development of calluses was negatively affected by the period of cold exposure. Calluses exposed to 16 days of cold exposure (Figure 2i) did not show discernible signs of growth, representing the most unfavorable performance in this context. In contrast, the control group exhibited stable growth (Figure 2j). It’s important to note that all treatments contained equivalent amounts of calluses distributed across the plates.
Double-stained embryogenic cultures after recovery from the cold treatments and growth at 25±2 °C in the dark. (a-b) two days cold treatment, (c-d) four days cold treatment, (e-f) eight days cold treatment, (g-h) sixteen days cold treatment. (i) Embryogenic culture from the sixteen days of cold treatment, after 20 days of recovery and growth at 25±2 °C in the dark. (j) Embryogenic culture from the control treatment and 20 days growth at 25±2 °C in the dark.
The development cycle of an A. angustifolia zygotic embryo, from pollen dispersion to seed maturation, takes approximately 19 to 24 months (Mantovani et al. 2004, Goeten et al. 2020). During this period, the embryo experiences a winter phase in its early growth and another during seed maturation, with mean temperatures below 10 °C (Mantovani et al. 2004). Zygotic embryos collected for somatic embryogenesis establishment in A. angustifolia have not gone through both winter seasons during their development. This study hypothesized that inducing this stress would activate a putative cold memory of the embryogenic masses and prompt somatic embryo maturation.
Seasonal signals play a crucial role in determining the timing of developmental changes in plants. In plants like Arabidopsis thaliana and overwintering crops such as wheat, specific genes are epigenetically regulated in response to cold periods, a process known as vernalization, which is necessary for flowering in spring (Baulcombe & Dean 2014). Cold temperatures in autumn lead to the suppression of the FLOWERING LOCUS C gene in Arabidopsis, while in winter, the VERNALIZATION INSENSITIVE3 gene is activated in response to prolonged cold and the absence of daily temperatures above 15°C (Hepworth et al. 2018). Similar genes in wheat are also regulated in this manner, allowing the plants to flower in late spring after a prolonged cold period in winter (Xu & Chong 2018, Niu et al. 2024). This epigenetic memory of winter, established through the cold-induced activation of the VERNALIZATION1 gene in wheat, is inherited by early embryos but reset in later stages of embryo development (Niu et al. 2024). This suggests that periods of low temperature are recognized during embryonic stages and influence important developmental processes in plants.
Exposing Acca sellowiana embryogenic cultures to cold treatment at 4.5 °C for up to 80 hours improved somatic embryo maturation (Guerra et al. 1997). However, keeping the embryogenic cultures at 4.0 °C for 20 days did not enhance the conversion of somatic embryos into plantlets for this species (Mengarda et al. 2009). In A. angustifolia, no improvement was observed even with shorter periods of cold treatment, and longer cold incubation periods led to smaller post-recovery culture growth. Researchers have extensively studied the obstacles of somatic embryogenesis in A. angustifolia using proteomic approaches (e.g. Silveira et al. 2008, Balbuena et al. 2011, Santos et al. 2016, Fraga et al. 2016), global DNA methylation (Fraga et al. 2015, 2016), and gene expression analyses (Schlögl et al. 2012a, b, Steiner et al. 2012). However, these studies, like the present one, did not conclusively identify the factors hindering the conversion of PEM III embryogenic masses of A. angustifolia into plantlets.
Even though our initial hypothesis about the induction of a cold memory in the embryogenic mass was refuted, we have gathered new information about epigenetic factors that could improve the further development of PEM III embryogenic masses in A. angustifolia. In short, our results show that subjecting the PEM III embryogenic cultures to cold treatments alone does not promote their further development and long in vitro cold periods may be harmful for the embryogenic mass. Therefore, addressing the bottlenecks in the A. angustifolia somatic embryogenesis protocol requires more integrative investigations. We recommend further studies on additional epigenetic controls of embryo maturation, with a focus on comparing zygotic and somatic embryo development. Currently, we are conducting a comprehensive analysis of miRNAs present at different stages of zygotic embryo development in A. angustifolia. This will help us understand the epigenetic control of this process and guide us in making adjustments to the culture media and the physical conditions of the in vitro culture.
ACKNOWLEDGMENTS
This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (Grant number 303673/2021-4 and PIBIC scholarship).
REFERENCES
- ASTARITA LV, FLOH EIS & HANDRO W. 2003. Changes in IAA, tryptophan, and activity of soluble peroxidase associated with zygotic embryogenesis in Araucaria angustifolia (Brazilian pine). Plant Growth Reg 39: 113-118.
- BALBUENA TS, JO L, PIERUZZI FP, DIAS LLC, SILVERIA V, SANTA-CATARINA C, JUNQUEIRA M, THELEN JJ, SHEVCHENCKO A & FLOH EIS. 2011. Phytochemistry differential proteome analysis of mature and germinated embryos of Araucaria angustifolia. Phytochem 72: 302-311.
- BAULCOMBE DC & DEAN C. 2014. Epigenetic regulation in plant responses to the environment. Cold Spring Harb Perspect Bio 6: a019471.
- FRAGA HP, VIEIRA LN, PUTTKAMMER CC, OLIVEIRA EM & GUERRA MP. 2015. Time-lapse cell tracking reveals morphohistological features in somatic embryogenesis of Araucaria angustifolia (Bert) O Kuntze. Trees 29: 1613-1623.
- FRAGA HPF, VIEIRA LN, PUTTKAMMER CC, SILVA JM, ANJOS KG, OLIVEIRA EM & GUERRA MP. 2016. High-efficiency cryopreservation of Araucaria angustifolia (Bertol.) Kuntze embryogenic cultures: ultrastructural characterization and morpho-physiological features. Plant Cell Tiss Organ Cult 124: 307
- GOETEN D, ROGGE-RENNER GD, SCHMIDT ÉC, BOUZON ZL, FARIAS-SOARES FL, GUERRA MP & STEINER N. 2020. Atualizando a ontogênese embrionária em Araucaria angustifolia : de Burlingame (1915) até o presente. Protoplasma 257: 931-948.
- GUERRA MP, PESCADOR R, DAL VESCO LL, NODARI RO & DUCROQUET JPHJ. 1997. In vitro morphogenesis in Feijoa sellowiana: somatic embryogenesis and plant regeneration. Acta Hortic 452: 27-36
- GUPTA PK & PULLMAN GS. 1991. Method for reproducing coniferous plants by somatic embryogenesis using abscisic acid and osmotic potential variation. - US patent No. 5,036,077.
- HEPWORTH J ET AL. 2018. Absence of warmth permits epigenetic memory of winter in Arabidopsis. Nat Commun 9: 639.
- MANTOVANI A, MORELLATO LPC & REIS MS. 2004. Fenologia reprodutiva e produção de sementes em Araucaria angustifolia (Bert.) O. Kuntze. Braz J Bot 27: 787-796.
- MENGARDA LHG, PESCADOR R, CHU EP & FIGUEIREDO-RIBEIRO RCL. 2009. Efeito do frio sobre os carboidratos solúveis em culturas embriogênicas de Acca sellowiana O. Berg (Myrtaceae). Rev Bras Bot 32: 307-317.
- NIU D, GAO Z, CUI B, ZHANG Y & HE Y. 2024. A molecular mechanism for embryonic resetting of winter memory and restoration of winter annual growth habit in wheat. Nat Plants 10: 37-52.
- SANTOS ALW, ELBL P, NAVARRO BV, OLIVEIRA LF, SALVATO F, BALBUENA TS & FLOH EIS. 2016. Quantitative proteomic analysis of Araucaria angustifolia (Bertol.) Kuntze cell lines with contrasting embryogenic potential. J Proteom 130: 180-189.
- SCHLÖGL PS, DOS SANTOS ALW, VIEIRA LN, FLOH EIS & GUERRA MP. 2012a. Gene expression during early somatic embryogenesis in Brazilian pine (Araucaria angustifolia (Bert) O. Ktze). Plant Cell Tiss Organ Cult 108: 173-180.
- SCHLÖGL PS, DOS SANTOS ALW, VIEIRA LN, FLOH EIS & GUERRA MP. 2012b. Cloning and expression of embryogenesis-regulating genes in Araucaria angustifolia (Bert.) O. Kuntze (Brazilian Pine). Gen Mol Biol 35: 172-181.
- SILVEIRA V, SANTA-CATARINA C, BALBUENA TS, MORAES FMS, RICART CAO, SOUSA MV, GUERRA MP, HANDRO W & FLOH EIS. 2008. Endogenous abscisic acid and protein contents during seed development of Araucaria angustifolia. Biol Plant 52: 101-104.
- SILVEIRA V, STEINER N, DOS SANTOS ALW, NODARI RO & GUERRA MP. 2002. Biotechnology tolls in Araucaria angustifolia conservation and improvement: inductive factors affecting SE. Crop Breed App Biotechnol 2: 463-470.
- STEFENON VM, REE JF, PINHEIRO MVM, GOETEN D, STEINER N & GUERRA MP. 2020. Advances and constraints in somatic embryogenesis of Araucaria angustifolia, Acca sellowiana, and Bactris gasipaes. Plant Cell Tiss Ogan Cult 143: 241-263.
- STEFENON VM, STEINER N, GUERRA MP & NODARI RO. 2009. Integrating approaches towards the conservation of forest genetic resources: a case study of Araucaria angustifolia. Biodiv Cons 18: 2433-2448.
- STEINER N, FARIAS-SOARES FL, SCHMIDT ÉC, PEREIRA ML, SCHEID B, ROGGE-RENNER GD & GUERRA MP. 2015. Toward establishing a morphological and ultrastructural characterization of proembryogenic masses and early somatic embryos of Araucaria angustifolia (Bert.) O. Kuntze. Protoplasma 253: 487-501.
- STEINER N, SANTA-CATARINA C, GUERRA M, CUTRI L, DORNELAS M & FLOH E. 2012. A gymnosperm homolog of SE RECEPTOR-LIKE KINASE-1 (SERK1) is expressed during SE. Plant Cell Tiss Ogan Cult 109: 41-50.
- XU S & CHONG K. 2018. Remembering winter through vernalization. Nat Plants 4: 997-1009.
