Genetic engineering in the oilseed crop camelina: establishment and application

Barno Ruzimurodovna Rezayeva

doi:10.15488/19350

Genetic engineering in the oilseed crop camelina: establishment and application

Barno Ruzimurodovna Rezayeva

Abteilung Reproduktion und Entwicklung

Publikation: Qualifikations-/Studienabschlussarbeit › Dissertation

Abstract

Die in vitro-Regeneration und Agrobakterien-vermittelte genetische Transformation sind essentielle Methoden der Pflanzen- und Züchtungsforschung. Die Anwendungsmöglichkeiten moderner biotechnologischer Methoden sind jedoch bei einigen Nutzpflanzen durch noch zu ineffiziente Protokolle limitiert. Davon betroffen ist insbesondere die Nutzung des erheblichen Potenzials der Genom-Editierung. Um diese Einschränkungen bei der zunehmende Bedeutung gewinnenden Ölsaat Leindotter (Camelina, Camelina sativa) zu überwinden, wurden unreife zygotische Embryonen für die in vitro-Regeneration verwendet, da davon auszugehen war, dass Gewebe dieser Ausgangsexplantate im Verlgeich zu herkömmlichen, ausdifferenzierten Pflanzenteilen eine höhere Totipotenz aufweisen. Nachdem die Bildung von Adventivsprossen und deren Weiterentwicklung zu Pflanzen unter Verwendung der experimentellen Akzession Cam139 tatsächlich mit hoher Effizienz erreicht worden war, wurden die dem zugrunde liegenden initialen Entwicklungsprozesse auf zellulärer Ebene anhand von mikroskopischen Präparaten untersucht und dabei ermittelt, dass die regenerativen Strukturen ihren Ursprung in den äußersten Schichten des Hypokotyls haben. Zudem ist es gelungen dieses für dikotyledone Pflanzen weitgehend neuartige Regenerationsprinzip auch für die aktuelle Camelina-Sorte Ligena anzuwenden. Das neu entwickelte Regenerationsprinzip wurde dazu genutzt, eine Methode der Agrobakterien-vermittelten Transformation für Camelina zu etablieren. Bei diesen experimentellen Arbeiten wurden mehrere Einflussfaktoren variiert, darunter die gezielte mechanische Verwundung, die Dauer der Vor- und Co-Kultur sowie die Konzentration des Selektionsagens Hygromycin. Auch diese Methode erwies sich für aktuelle Sorten anwendbar, wobei Transformationsraten von bis zu 17 % (bezogen auf die Anzahl kultivierter Explantate) erreicht wurden. Schließlich wurde die neu etablierte Transformationsmethode verwendet, um das Gen des Fettsäure-modifizierenden Enzyms FATTY ACID ELONGASE 1 (FAE1) durch genspezifische Mutagenese mittels CRISPR-assoziierter (Cas)9-Endonuklease auszuschalten. Dazu wurde eine protoplastenbasierte Methode der funktionellen Validierung von in silico vorselektierten Cas9-Zielmotiven und den entsprechenden guide (g)RNAs entwickelt. Anhand dieses Tests wurden Mutationen in allen drei vorläufig ausgewählten, FAE1-spezifischen Zielmotiven aller drei homöologen Camelina-Subgenome nachgewiesen. Bei der Tiefensequenzierung Zielregion-spezifischer PCR-Amplicons von genomischer Protoplasten-DNA waren dabei bis zu 15 % der Sequenzier-Reads mutiert. Nach Verwendung des gleichen Konstrukts für die Herstellung stabil transgener Pflanzen wurden Mutationsereignisse ebenfalls in allen drei FAE1-spezifischen Zielmotiven aller drei Homöoallele nachgewiesen. Aufgrund dieser hohen Effizienz wiesen 8 von 9 primär-transgenen Pflanzen Mutationen in allen drei FAE1-Homöoallelen auf. Folglich konnten dreifach-homozygote fae1-Knockout-Mutanten bereits in der ersten Selbstungsgeneration identifiziert werden. Fettsäure-Profilanalysen dieser Knockout-Linien ergaben, dass die ernährungsphysiologisch besonders wertvollen Anteile von Linolsäure (C18:1) und -Linolensäure (C18:2) signifikant erhöht und die zu beeinträchtigter Ölqualität führende Erucasäure (C22:1) auf kaum detektierbare Spuren reduziert war. Insgesamt gesehen umfasst die vorliegende Arbeit bedeutende Fortschritte von der biotechnologischen Erschließung der Ölsaat Camelina bis hin zum erfolgreichen Einsatz der Genom-Editierung mit dem Ergebnis der Entwicklung von Elite-Linien mit bislang unerreicht hoher Ölqualität als wichtige Grundlage einer gesunden Ernährung.

Originalsprache	Englisch
Qualifikation	Doktor(in) der Naturwissenschaften (Dr. rer. nat.)
Gradverleihende Hochschule	Leibniz Universität Hannover
Betreuerin / Beraterin	Winkelmann, Traud, Betreuer*in
Datum der Bewilligung	7 Apr. 2025
Erscheinungsort	Hannover
Herausgeber (Verlag)	Leibniz Universität Hannover
DOIs	https://doi.org/10.15488/19350
Publikationsstatus	Veröffentlicht - 30 Juli 2025

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10.15488/19350Lizenz: CC BY 3.0 DE

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@phdthesis{dcf38a72062d4415a91893821cd6eccf,

title = "Genetic engineering in the oilseed crop camelina: establishment and application",

abstract = "In vitro regeneration and Agrobacterium-mediated genetic transformation are essential methods in plant and breeding research. However, the opportunities for applying modern biotechnological methods to some crops are limited by protocols that are still too inefficient. This particularly affects the utilization of the considerable potential of genome editing. To overcome these limitations in the increasingly important oilseed camelina (Camelina sativa), immature zygotic embryos were used for in vitro regeneration, as it was assumed that tissue from this explant type has a higher totipotency compared to conventionally used, more differentiated plant parts. After the formation of adventitious shoots and their further development into plants using the experimental accession Cam139 had indeed been achieved with high efficiency, the underlying initial developmental processes were investigated at the cellular level using microscopic preparations, and it was determined that the regenerative structures originate in the outermost layers of the embryonic hypocotyl. In addition, this regeneration principle, which is largely unexplored for dicotyledonous plants, has also been successfully applied to the current camelina variety Ligena. The newly developed regeneration method was then used to establish Agrobacteriummediated transformation for camelina. Several influencing factors were varied in this experimental work, including the targeted mechanical wounding, the duration of the pre- and co-culture and the concentration of the selection agent hygromycin. This method also proved to be applicable to current varieties, with transformation efficiencies of up to 17\% (based on the number of cultivated explants) being achieved. Finally, the newly established transformation method was used to knock out the gene encoding the fatty acid-modifying enzyme FATTY ACID ELONGASE 1 (FAE1) by targeted mutagenesis using CRISPR-associated (Cas) 9 endonuclease. To this end, a protoplast-based method of functional validation of in silico pre-selected Cas9 target motifs and their cognate guide (g)RNAs was developed. Using this test, mutations were detected in all three preselected FAE1-specific target motifs of all three homeologous camelina subgenomes. In the deep-sequencing of target region-specific PCR amplicons of genomic protoplast DNA, up to 15\% of the sequencing reads proved mutated. After using the same construct to produce stably transgenic plants, mutation events were also detected in all three FAE1-specific target motifs of all three homeoalleles. Thanks to this high efficiency, 8 out of 9 primary transgenic plants carried mutations in all three FAE1 homeoalleles. Consequently, triple-homozygous fae1 knockout mutants could already be identified in the first selfing generation. The fatty acid profiles of these knockout lines showed that the nutritionally particularly valuable proportions of linoleic acid (C18:1) and a-linolenic acid (C18:2) were significantly increased and that erucic acid (C22:1), which leads to impaired oil quality, was reduced to barely detectable traces. Overall, this work comprises significant advances from a basic biotechnological exploration of the oilseed camelina to the successful use of genome editing, resulting in the development of elite lines with unprecedented high oil quality as an important prerequisite for a healthy diet.",

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AU - Rezayeva, Barno Ruzimurodovna

PY - 2025/7/30

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N2 - In vitro regeneration and Agrobacterium-mediated genetic transformation are essential methods in plant and breeding research. However, the opportunities for applying modern biotechnological methods to some crops are limited by protocols that are still too inefficient. This particularly affects the utilization of the considerable potential of genome editing. To overcome these limitations in the increasingly important oilseed camelina (Camelina sativa), immature zygotic embryos were used for in vitro regeneration, as it was assumed that tissue from this explant type has a higher totipotency compared to conventionally used, more differentiated plant parts. After the formation of adventitious shoots and their further development into plants using the experimental accession Cam139 had indeed been achieved with high efficiency, the underlying initial developmental processes were investigated at the cellular level using microscopic preparations, and it was determined that the regenerative structures originate in the outermost layers of the embryonic hypocotyl. In addition, this regeneration principle, which is largely unexplored for dicotyledonous plants, has also been successfully applied to the current camelina variety Ligena. The newly developed regeneration method was then used to establish Agrobacteriummediated transformation for camelina. Several influencing factors were varied in this experimental work, including the targeted mechanical wounding, the duration of the pre- and co-culture and the concentration of the selection agent hygromycin. This method also proved to be applicable to current varieties, with transformation efficiencies of up to 17% (based on the number of cultivated explants) being achieved. Finally, the newly established transformation method was used to knock out the gene encoding the fatty acid-modifying enzyme FATTY ACID ELONGASE 1 (FAE1) by targeted mutagenesis using CRISPR-associated (Cas) 9 endonuclease. To this end, a protoplast-based method of functional validation of in silico pre-selected Cas9 target motifs and their cognate guide (g)RNAs was developed. Using this test, mutations were detected in all three preselected FAE1-specific target motifs of all three homeologous camelina subgenomes. In the deep-sequencing of target region-specific PCR amplicons of genomic protoplast DNA, up to 15% of the sequencing reads proved mutated. After using the same construct to produce stably transgenic plants, mutation events were also detected in all three FAE1-specific target motifs of all three homeoalleles. Thanks to this high efficiency, 8 out of 9 primary transgenic plants carried mutations in all three FAE1 homeoalleles. Consequently, triple-homozygous fae1 knockout mutants could already be identified in the first selfing generation. The fatty acid profiles of these knockout lines showed that the nutritionally particularly valuable proportions of linoleic acid (C18:1) and a-linolenic acid (C18:2) were significantly increased and that erucic acid (C22:1), which leads to impaired oil quality, was reduced to barely detectable traces. Overall, this work comprises significant advances from a basic biotechnological exploration of the oilseed camelina to the successful use of genome editing, resulting in the development of elite lines with unprecedented high oil quality as an important prerequisite for a healthy diet.

AB - In vitro regeneration and Agrobacterium-mediated genetic transformation are essential methods in plant and breeding research. However, the opportunities for applying modern biotechnological methods to some crops are limited by protocols that are still too inefficient. This particularly affects the utilization of the considerable potential of genome editing. To overcome these limitations in the increasingly important oilseed camelina (Camelina sativa), immature zygotic embryos were used for in vitro regeneration, as it was assumed that tissue from this explant type has a higher totipotency compared to conventionally used, more differentiated plant parts. After the formation of adventitious shoots and their further development into plants using the experimental accession Cam139 had indeed been achieved with high efficiency, the underlying initial developmental processes were investigated at the cellular level using microscopic preparations, and it was determined that the regenerative structures originate in the outermost layers of the embryonic hypocotyl. In addition, this regeneration principle, which is largely unexplored for dicotyledonous plants, has also been successfully applied to the current camelina variety Ligena. The newly developed regeneration method was then used to establish Agrobacteriummediated transformation for camelina. Several influencing factors were varied in this experimental work, including the targeted mechanical wounding, the duration of the pre- and co-culture and the concentration of the selection agent hygromycin. This method also proved to be applicable to current varieties, with transformation efficiencies of up to 17% (based on the number of cultivated explants) being achieved. Finally, the newly established transformation method was used to knock out the gene encoding the fatty acid-modifying enzyme FATTY ACID ELONGASE 1 (FAE1) by targeted mutagenesis using CRISPR-associated (Cas) 9 endonuclease. To this end, a protoplast-based method of functional validation of in silico pre-selected Cas9 target motifs and their cognate guide (g)RNAs was developed. Using this test, mutations were detected in all three preselected FAE1-specific target motifs of all three homeologous camelina subgenomes. In the deep-sequencing of target region-specific PCR amplicons of genomic protoplast DNA, up to 15% of the sequencing reads proved mutated. After using the same construct to produce stably transgenic plants, mutation events were also detected in all three FAE1-specific target motifs of all three homeoalleles. Thanks to this high efficiency, 8 out of 9 primary transgenic plants carried mutations in all three FAE1 homeoalleles. Consequently, triple-homozygous fae1 knockout mutants could already be identified in the first selfing generation. The fatty acid profiles of these knockout lines showed that the nutritionally particularly valuable proportions of linoleic acid (C18:1) and a-linolenic acid (C18:2) were significantly increased and that erucic acid (C22:1), which leads to impaired oil quality, was reduced to barely detectable traces. Overall, this work comprises significant advances from a basic biotechnological exploration of the oilseed camelina to the successful use of genome editing, resulting in the development of elite lines with unprecedented high oil quality as an important prerequisite for a healthy diet.

U2 - 10.15488/19350

DO - 10.15488/19350

M3 - Doctoral thesis

PB - Leibniz Universität Hannover

CY - Hannover

ER -