Enhancing the Efficiency of Somatic Embryogenesis and Genetic Transformation in Theobroma cacao L.
Restricted (Penn State Only)
- Author:
- Bhatt, Jishnu
- Graduate Program:
- Plant Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 20, 2024
- Committee Members:
- Teh-Hui Kao, Program Head/Chair
Siela Maximova, Chair & Dissertation Advisor
Howard Salis, Outside Field Member
Mark Guiltinan, Major Field Member
Teh-Hui Kao, Outside Unit Member - Keywords:
- Plant
Transformation
Somatic Embryo
Biotechnology
Binary Vector
Cacao
Plant Tissue Culture
Genetic Engineering - Abstract:
- The chocolate tree, Theobroma cacao L. or cacao, is one of the most globally important woody tree species because its seeds provide the raw ingredients to make chocolate. Global cacao productivity is threatened by diseases and pests which result in annual potential losses of around thirty percent while its demand is ever-growing. Currently, there is a global replanting requirement of over one billion trees annually that is not being met. Utilizing high efficiency propagation systems such as somatic embryogenesis (SE) and the acceleration of genetic improvements for disease resistance through biotechnology are powerful strategies to combat these problems. Both basic and applied research in cacao is currently bottlenecked by limitations in the regeneration of somatic embryos (SEs) and the genetic modification system, highlighting the importance of improving these methodologies. The work in this dissertation begins with investigating a means to expand the tissue range by which SE can be initiated from the cacao tree. Nearly all tissues in cacao are recalcitrant to the formation of embryogenic calli, from which SEs are indirectly regenerated. Initiating embryogenic cultures has only been possible from petals and staminodes of immature cacao flowers, or zygotic tissues as starting explants and limits all SE work to adult, flowering trees. Previous work has established that the overexpression of the Leafy Cotyledon 2 (TcLEC2) gene significantly enhanced the embryogenic potential of floral explants, and its activation allowed for regeneration of SEs from cacao leaves. In Chapter 2, a tissue from transgenic tree expressing inducible TcLEC2-glucorticoid receptor fusion protein was used to evaluate embryogenic potential of additional vegetative tissues. Upon activating TcLEC2 for a 72-hour pulse, by adding dexamethasone to the regeneration medium, all tissues including peduncles, shoot apices, petioles, internodes, and leaves from development stages A-D were able to regenerate SEs. Notably, under the induction conditions, explants from fully expanded leaves (developmental stage C and D) regenerated SEs at rates similar to floral petals. In addition, the leaves regenerated abnormal SEs at lower rates compared to other vegetative tissues evaluated in this experiment. This demonstrates that various non-sexual somatic tissues from juvenile cacao trees contain cells that are competent for embryonic conversion. Most plant genetic engineering methods are based on Agrobacterium tumefaciens-mediated transformation. A. tumefaciens excises and delivers T-DNA (transfer DNA) molecules often from a binary vector (or plasmid) to be incorporated into the genome of plant cells through a process called genetic transformation. It is well known that the compatibility of the plant host and A. tumefaciens strain are important factors for transformation success. However, it has not been well reported in the literature the influence of the binary vector and/or T-DNA design on transformation success. In Chapter 3, three binary vectors were functionally tested using a cacao leaf transient expression assay to determine whether vector backbone could aid in overcoming transformation recalcitrance. It was found that a ‘pLSU’ vector backbone outperformed the others by transiently expressing an enhanced green fluorescent protein (EGFP) reporter gene by over 18% and therefore was chosen for further optimization experiments. The T-DNA region of the pLSU binary vector was re-designed to be compatible with restriction enzyme-based, recombinase-based, PCR-based and Golden-Gate based cloning methods and additionally modified to contain translationally efficient Kozak sequences. The modified pLSU binary vector with the improved T-DNA region was used for a stable transformation of cacao SE cotyledons. Compared to the standard pCambia-based binary plasmid, pLSU resulted in a 3.49-fold increase in EGFP fluorescence in transformed cells measured six days after transformation. However, under the experimental conditions provided, transient transformation coverage and regeneration efficiency of stable transgenics was not improved, indicating that further optimization is required to overcome stable transformation recalcitrance, which is bottlenecked by other factors such as the host plants biological variability. This work demonstrates that ectopically activating the TcLEC2-GR fusion protein contributed to in vitro regeneration of SEs from previously recalcitrant somatic tissues of cacao (Chapter 2). TcLEC2 is one of a growing number of developmental regulating genes (DRGs) that have been demonstrated to have embryogenesis-promoting effect. In Chapter 4 of this work, five additional DRGs SERK1, PLT5, WIND1, WUS, and WOX9 were chosen based on their potential to improve SE in cacao and to be used as positive selection for regeneration of transgenic plants. The ortholog genes were identified in the cacao Scavina 6 genome bioinformatically, synthesized and cloned into the improved binary vectors described in Chapter 3 under control of an estradiol-inducible system (XVE-DRGs). The estradiol-inducible system was tested using an EGFP transient expression assay in tobacco leaves and was validated to be functional and ‘non-leaky.’ The five XVE-DRG vectors were transformed into wild-type PSU-Sca6 cacao secondary SE (SSE) cotyledons using a standard cacao transformation protocol. DRG expression was induced one month after culture initiation (ACI) in vitro. Approximately 400 explants were imaged individually at 7 time points over a three-month period under a fluorescence microscope to quantify regeneration and transformation metrics and the influence of DRG expression. It was found that a two-week pulse of DRG induction one-month ACI was not effective at changing the growth rate of calli or the regeneration of both non-transgenic and transgenic tertiary SEs (TSEs). Analysis of EGFP fluorescence as a marker for successful transformation revealed that stable transformation area coverage of cacao SSE cotyledon explants averaged around 9.5% and cacao calli grew at a rate of ~4.4% per week. Additionally, the size of SSE cotyledon explants did not show correlation with their transformation coverage and TSE regeneration rate. The growth rate of calli was analyzed under increasing concentrations of geneticin antibiotic, and the results showed that increasing the concentration to 150 mg/L was inhibitory for both non-transgenic and transgenic cacao cells in vitro, establishing a previously unreported upper threshold for geneticin selection. This study also yielded six transgenic TSEs from transformations of all the XVE-DRGs except TcWUS, which can be utilized for further investigations into their potential for overcoming cacao in vitro and transformation recalcitrance. This work aimed to improve the efficiency of regeneration of genetically modified cacao somatic embryos for basic and applied plant science research. First, it demonstrated that nearly all differentiated non-sexual somatic cells of the juvenile cacao tree must harbor pre-competent and/or competent cells which require the correct spatiotemporal ectopic expression of totipotency-promoting genes for their conversion towards an embryogenic fate. Next, it provided evidence that the binary vector backbone is an important element of transformation recalcitrance. Finally, methodology was created to characterize transformation and in vitro regeneration in cacao to generate new insights on how the system can be optimized in the future. Altogether these findings are synergistic towards improving the transformation and regeneration system of cacao and identified knowledge gaps that must be addressed to overcome the recalcitrance of this globally important and beloved tree.