CYBRIDIZATION: A NEW BIOTECHNOLOGICAL TOOL FOR CITRUS IMPROVEMENT

This proposal will focus in the first stage on the evaluation of our new grapefruit cybrids and subsequently provides the industry with the needed grapefruit cultivar to revive and sustain the grapefruit industry in the era of citrus canker and HLB. Simultaneously, we will continue to produce more grapefruit and sweet orange cybrids especially from commercially important cultivars. We are proposing here to evaluate new grapefruit cybrid plants for enhanced citrus canker and HLB tolerance under challenging field conditions.

Objective 1: Production, validation and evaluation of new different sweet orange and grapefruit cybridsProduce, validate and evaluate of grapefruit somatic cybrids by the fusion of protoplasts derived from embryogenic callus cultures of 'Meiwa' kumquat, highly resistance to citrus canker, or using embryogenic callus from other canker resistant parents such as calamondin and papeda with leaf-derived protoplasts of the different highly susceptible grapefruit cultivars, mainly high-quality grapefruit and HLB tolerance if possible. Grapefruit cultivars that will be used to produce cybrid include but not limited to red grapefruit (e.g. N40-16-11-11, N11-17, N11-29, N40-16-11-15, and N40-16-11-3). Sweet orange cultivars that will be used to produce cybrid include but not limited to EV1, EV2, OLL6, OLL7, and Vernia.Objective 2.: Evaluation of growth rate and citrus canker tolerance in grapefruit cybrids by measuring growth rate parameters and monitoring citrus canker symptom development (lesion size and number).Citrus canker symptom development (lesion size and number) will assessed according standard methods. Bacterial populations will be determined on selected genotypes in the field according to Murata et al. (2019).Tree size will be assessed at the start of the project and in July/December every year by measuring trunk diameter (5 cm above the bud union), tree height to top of canopy (not including height of vigorous shoots that extend significantly past the top of the canopy), and canopy diameter (in parallel and perpendicular to the tree row). Canopy volume will be calculated using the formula: [(diameter parallel to row × diameter perpendicular to row) × height]/4. If the plants start flowering, blooming period will be evaluated.Canopy thickness and color ratings will be scaled from 1-5 as follows:Canopy thickness: 1 = very thin canopy, 2 = thin canopy, 3 = medium canopy, 4 = thick canopy, 5 = very thick canopy (typical of pre-HLB tree).Objective 3: Evaluation of HLB tolerance in grapefruit cybrids by monitoring CLas titers using qRT-PCR, a disease health index, and tree growth.HLB diagnosis of mature leaves for Candidatus Liberibacter asiaticus (CLas) titer and activity will be measured annually by using the qRT-PCR (Li et al. 2006). A total of 4-6 mature leaves will be collected from December to April in each of all four cardinal sections per tree, from which the petiole/midribs will be used for CLas detection.Objective 4: Determine leaf and soil nutrient concentrations for fertilization adjustment, optimized pH and nutrition.Leaf and soil nutrient concentrations will be determined annually in July/August (on spring flush) for fertilization adjustments. For that reason, we are not sampling all cybrids. Twenty-thirty mature leaves will be collected in different parts of the tree. Roots will be collected on 2 sides perpendicular to the tree row. Leaf and root tissue samples will be dried for 72 h at 65oC and analyzed by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) (Obreza and Morgan 2008).Objective 5: Evaluate yield and fruit quality when possible (includes brix, acid, brix/acid ratio, internal and external color, and fruit size).For the new grapefruit cybrids, if and when the trees produce fruit a sample of harvested fruit will be measured for size (fresh wt, diameter, % juice content), peel color, citric acid content, and percent juice content each season, including the relative amounts of "good" vs "greening" fruit, and report as preliminary data. Yield per tree and per acre, fruit weight, fruit size, and fruit quality will be determined annually at harvest.Objective 6: To understand how primary metabolism is affected by cybridization in grapefruit, by studying the comparative responses of different clear/mitochondrion/chloroplast combinations during citrus canker disease development.For the purpose of the current proposal, we will choose four different genotypes; 1) 'Meiwa' kumquat as an embryogenic parent, 2) 'Marsh' grapefruit as mesophyll parent, 3) Marsh cybrid that contains both kumquat mitochondria and chloroplast, and 4) Marsh cybrid that contains kumquat mitochondria and grapefruit chloroplast. The selected plants will be multiplied by grafting onto a selected rootstock. All plants will be grown under the same greenhouse conditions and compared in terms of the development of the Xcc bacterium, which causes citrus canker disease. All plant manipulations will be performed at the facilities of the Citrus Research and Education Center (CREC), IFAS, University of Florida, Lake Alfred, FL.Objective 7: To improve our understanding of the natural mechanisms of the chloroplast genome in plant defense by studying some related disease resistance gene expression in the field trees; this will subsequently help to design new varieties and/or genetically modified existing susceptible cultivars for tolerance/resistance against citrus canker and HLB.This part will be done by looking at specific disease resistance genes expression levels as an outcome of cytoplasm and nuclear genome interactions in the various combinations. Data generated from gene expression study and metabolic profile will be analyzed using specific software to study the change in the metabolic pathway in different cybrid combination as a result of cytoplasm/nuclear genome interaction.