Gene editing-assisted generation of delayed bolting lettuce (Lactuca sativa L.) for high temperature conditions
Meghan Roche, PhD Final Seminar
Tuesday, July 30, 2024, 10 am
(Under the direction of Dr. Ricardo Hernandez and Dr. Wusheng Liu, Co-Chairs)

Location: 121 Kilgore / Hybrid
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https://ncsu.zoom.us/j/91000712050?pwd=cIhOINNfIddEUhhBEtrVXjMINwxcvc.1
Meeting ID: 910 0071 2050
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 Roche.Abstr.pdf

Abstract

Lettuce (Lactuca sativa L.) is a popular crop for cultivation and consumption, with the farming of lettuce increasing in popularity in recent years. With increased interest in this crop, growers are expanding from fields into new cultivation systems, including indoor controlled-environment methods, such as greenhouses and vertical farms. Lettuce cultivation does not come without challenges, however, as increasing global temperatures push demand for cultivars with improved stress adaptations, and yield demands in indoor cultivation favor enhanced growing temperatures, in spite of the threat of premature bolting. Bolting, the transition from vegetative to reproductive growth, reduces quality and makes the lettuce head unsalable, causing economic loss to growers. Thus, while climate change necessitates the development of crops adapted to warmer temperatures for field cultivation, so too can the breeding of crops for controlled-environment cultivation. Towards this aim, the primary objective of this work was to knockout floral promotion genes in lettuce to delay the onset of heat-induced bolting. To achieve this objective, two goals had to first be achieved; optimization of a lettuce transformation protocol in the cultivars of interest, and identifying the genes to target for knockout in lettuce via thorough literature review.

In spite of the achievements in lettuce transformation and regeneration, there remains a genotype dependence upon the ability to efficiently transform and regenerate lettuce plants. In this work, an optimized Agrobacterium-mediated transformation protocol was successfully developed for elite lettuce cultivars of the romaine, leaf, and butterhead cultivar types. A plant growth regulator combination of 1-naphthaleneacetic acid (NAA; 0.10 mg/L) and 6-benzyladenine (BA; 0.25 mg/L), the use of cotyledons and the first true leaf as explants, and the use of hygromycin (15 mg/L) for transgenic plant selection worked well for seven out of the eleven tested cultivars, achieving a 24.3% – 100% transformation efficiency. These seven cultivars include two romaine-types, three leaf-types, and two butterhead-types, and marks the first successful genetic transformation of the romaine cvs. ‘Kahu’ and ‘Rosalita’, the leaf lettuces ‘Red Sails’ and ‘Royal Oak Leaf’, and the butterhead ‘Lollo Biondo’. While our optimized protocol did not successfully regenerate plants in the remaining four cultivars, successive use of kanamycin selection (40 mg/L) with the same plant growth regulators enabled transformation in the butterhead-type ‘Mariska’.

Flowering regulation in the model species Arabidopsis thaliana involves five defined floral induction pathways; autonomous, vernalization, photoperiod, gibberellin, and thermosensory. In the complex regulation of floral timing, no pathway exists exclusively of another, as components are frequently observed to overlap and/or exhibit functionality in multiple pathways at once. While the genes of the autonomous pathway were once thought to contribute to flowering regardless of environmental conditions, convergence between autonomous pathway factors and temperature-based flowering led to the identification of the thermosensory pathway. Accordingly, the key autonomous pathway genes serve crossover function in both pathways. In the thermosensory pathway, warm temperatures trigger the acceleration of flowering, and autonomous pathway genes are central in coordinating these environmental responses. From this deep dive into Arabidopsis flowering pathways, and study of their conserved function in Asteraceae, three candidate genes were identified for knockout in lettuce, FLOWERING LOCUS T (FT), FLOWERING LOCUS CA (FCA), and FLOWERING LOCUS D (FLD), all identified to play integral roles in the warm temperature induced initiation of flowering.

CRISPR/Cas9 was utilized to target the endogenous lettuce genes, LsFT, LsFCA and LsFLD for editing, with the goal of delaying bolting in warm environments. While bona fide editing of the target genes could not be confirmed, this effort produced lines with marked delays in lettuce bolting when cultivated under increased ambient temperatures, demonstrating its potential in future grower and breeder applications.