REGENERATION OF BLUEBERRY (VACCINIUM SPP.)

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0224919
• Project Start Date: Jan 1, 2011
• Project End Date: Dec 31, 2015
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF DELAWARE
(N/A)
NEWARK,DE 19717

Performing Department
Plant & Soil Sciences

Non Technical Summary
Non-technical summary: Presently, it is difficult to transform blueberry due to the cells within the leaf explant that are capable of being transformed and regenerated. The development of a regeneration system that relied on callus might improve transformation success. While transformation of blueberry has been reported, subsequent publications have not been forthcoming confirming the transformation-regeneration recalcitrance of blueberry. An alternative regeneration system via callus might improve blueberry's transformation frequency.

Animal Health Component

30%

Research Effort Categories

Basic

70%

Applied

30%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
205	1120	1060	50%
206	1120	1060	50%

Knowledge Area
206 - Basic Plant Biology; 205 - Plant Management Systems;

Subject Of Investigation
1120 - Blueberry;

Field Of Science
1060 - Biology (whole systems);

Keywords

blueberry

regeneration

callus

indirect

transformation

ericaceae

Goals / Objectives
I want to continue my 26 plus years of work with plants native to the eastern U.S.; however, I want to shift my focus from herbaceous to woody perennials. I am interested in working with Vaccinium spp. (specifically, blueberry). I want to develop protocols for regenerating indirectly through callus preferably via somatic embryos. While blueberry is presently regenerated through direct organogenesis from leaf culture, production of transformants is limited due to lack of ease of regeneration. The purpose of developing an indirect regeneration procedure (that is, through callus) is to improve/increase regeneration rates especially in some economically important cultivars that historically have been less responsive in vitro (e.g., 'Bluecrop'). Development of a callus-based, indirect regeneration protocol may allow for, by extension, the increased generation of transformants. Improved/increased production of transformants may help with the elucidation of genes specific to the improvement of blueberry.

Project Methods
Plant material: Blueberry cultivars will be selected based on their commercial importance and their ease of culture. Initially, cultivars will include 'Bluecrop', 'Duke', 'Elliott', 'Aurora' 'Legacy', 'Brightwell', and 'Star' (presently in culture). Experiments will use cultivars that are generally recognized as being more responsive in vitro (e.g., 'Aurora') with the thought that once developed, the regeneration protocols will be transferred to more commercially important cultivars (e.g., 'Bluecrop'). Regeneration: Factors such as origin of explant, medium constituents, and environment will be investigated. Experimental components will be varied as needed based on work within the Ericaceae. Explants will include those that have been indirectly regenerative such as nodes, leaves, and flower bud pedicels. Medium constituents that have proven to be critical to regeneration in the Ericaceae have included salt formulation; growth regulator type, concentration, ratio and regime, organic additives and gelling agent. 'More active' auxins [pichloram and dicamba] as well as additional addenda (e.g., coconut water) also will be tested. Environmental conditions to be examined will include light vs. dark. Experiments will be maintained at 24 percent C and subcultured every 3 - 4 wk. All heat labile components will be filter sterilized and added to cooled, autoclaved media. Approach: There will be no exact sequence of experiments. Initial experiments will use leaves (easily available from in vitro-maintained stock cultures) that will be cultured on media containing cytokinins and auxins at typical concentrations (ca. 5 micro M). Cultures will initially be scored for those treatments that generate callus (Personal experience has demonstrated that blueberries can be challenging when it comes to generating callus.) Once growth regulators are identified to which blueberry is responsive, protocol refinement will occur (e.g., medium additives will be introduced with the objective of generating callus with regenerative capability). Micropropagation: The resultant shoots will be micropropagated on the following medium: WPM salts (Lloyd and McCown, 1980), MS vitamins (Murashige and Skoog, 1962), 2 percent (w/v) sucrose, 2 mg/l (9.12 micro M) zeatin, 556 mg/l Ca(NO3)2*4H20,and 5 gm/l Agargel. Media pH will be 5.2. Stocks will be subcultured every 3 wk. There are no reported difficulties for rooting blueberry microcuttings whether in vitro or ex vitro; therefore, standard procedures will be used. Experimental design and data analysis: Experiments will be designed as completely randomized blocks. Regeneration (callus generation, callus type, explant regenerability, number of regenerants/explant, and rate of regeneration) data will be quantified from all experiments. Experiments will be repeated at least once and treatments will have at least 20 units so that data can be analyzed with SAS procedures GLM, ANOVA, and REGRESS and means separated with Tukey's Studentized test where appropriate.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? The callus that has been generated will be maintained while at the same time it will be used to set up embryo-initiation experiments. Maintenance and continued growth of the organized ‘clumps will be attempted. New experiments looking at callus initiation and maintenance will be explored.

Impacts
What was accomplished under these goals? This project has been in progress for 30 months. Leaves of blueberry (Vaccinium) cultivars ‘Elliott’ and ‘Bluecrop’ have been cultured adaxial surface in contact with the medium. Three experimental avenues have been explored this period: basal salt formulation, sucrose concentration, and replication of experiments purported to generate embryos by Ericaceous plants. Experiments were placed in the dark unless otherwise noted. Basal salt formulation (MS [control], ½ MS, WPM, SH, G5) had no improved effect on callus initiation, calli from all treatments were very hard and non-friable. Leaves cultured on medium containing 6 percent sucrose generated more callus compared to medium containing 3 percent sucrose. ‘Elliott’ is a better model plant compared to ‘Bluecrop’; ‘Bluecrop’ has been very recalcitrant with regard to generating callus. All future work was carried out using ‘Elliott’. Experimental protocols purported to generate embryos from leaves of Ericaceous plants [blueberry (personal communication), Conostephium pendulum (Anthony et al., 2004b), Leucopogon verticillatus (Anthony et al., 2004a) were examined. None of these three protocols generated embryos from ‘Elliott’ leaves; however, Leucopogon-medium cultured leaves generated many organized clumps. These clumps were subcultured onto various treatments to see if they would self-replicate. Subculture of the clumps to Conostephium medium resulted in shoot elongation, subculture to Leucopogon medium resulted in the generation of more clumps, and subculture to callus maintenance medium resulted in browning. Callus (maintained under 10 micromoles per meter squared per second of PAR) that was initiated in early 2013 is friable, greening, and sufficiently massed up for use in embryo initiation experiments. A manuscript identifying the cells responsible for and the sequence of events leading up to shoot organogenesis is in review. Cells responsible for shoot regeneration from leaves have been identified as… Anthony, J.M., T. Senaratna, K.W. Dixon, and K. Sivasithamparam. 2004a. Somatic embryogenesis for mass propagation of Ericaeae – a case study with Leucopogon verticillatus. Plant Cell Tiss. Org. Cult. 76:137-146. Anthony, J.M., T. Senaratna, K.W. Dixon, and I. Sivasithamparam. 2004b. The role of antioxidants for initiation of somatic embryos with Conostephium pendulum (Ericaceae). Plant Cell, Tiss. Org. Cult. 78:247-252.

Publications

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: To date, there have been no outputs to share. PARTICIPANTS: Sherry Kitto worked on this project alone. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This project has been in progress for 18 months. The initial approach has been to 1) generate callus using indirect-regeneration protocols reported to be successful with relatives of blueberry (Vaccinium) and 2) use blueberry cultivars that have importance in the trade. So, leaves of blueberry cultivar Elliott have been cultured on Murashige-Skoog basal medium supplemented with (in mg per liter) 0.4 thiamine HCl, 0.5 pyridoxine HCl, 0.5 nicotinic acid, 100 myo-inositol, 60,000 sucrose, 40 adenine sulfate, and 100 casein hydrolysate. The pH was adjusted to 5.2 and 6 gm per liter agar (Sigma) added prior to autoclaving. Cultures were placed under 10 micromoles per meter squared per second of PAR. Callus has been subcultured monthly in an effort to establish leaf-free callus cultures. Leaves were cultured adaxial surface in contact with the medium. Two experimental avenues have been explored this period, coconut milk and cytokinin type. 1) Coconut milk. Basal medium containing 10 micromoles of thidiazuron (TDZ) was supplemented with 50%, 25%, 12.5%, or 6% deproteinized coconut milk (v/v). Six percent coconut milk generated the most and best looking (friable, creamy colored) callus. 2) Cytokinin type. Leaves were cultured on basal medium containing 6% (v/v) deproteinized coconut water supplemented with the following cytokinins: benzyl adenine, TDZ, kinetin, or 2iP. Leaves turned brown on benzyl adenine and generated no callus. Leaves on kinetin remained green but failed to generate callus. Callus was generated on the control (no cytokinin), the TDZ and the 2iP treatments. Some callus generated on TDZ was creamy in color and friable. The majority of the callus from the control and 2iP treatments was brown. After four weeks, TDZ treatments had substantial shoot regeneration (direct from the explant); however, after subculture and four additional weeks of growth, no regenerants were visible. While the 2iP-treated leaves had very little regeneration visible after four weeks, after subculture and four additional weeks of growth, shoot regeneration was increased. Blueberry leaves were slow to produce callus and several subcultures are required to mass it up.

Publications

• No publications reported this period

Progress 07/12/11 to 12/31/11

Outputs
OUTPUTS: This project has been in progress for six months. The initial approach has been to 1) generate callus using indirect-regeneration protocols reported to be successful with relatives of blueberry (Vaccinium) and 2) use blueberry cultivars that have important in the trade. So, leaves of blueberry cultivar Elliott have been cultured on Murashige-Skoog based media supplemented with (in mg per liter) 1.25-20 thidiazuron, 0.4 thiamine HCl, 0.5 pyridoxine HCl, 0.5 nicotinic acid, 100 myo-inositol, 60,000 sucrose, 40 adenine sulfate, and 100 casein hydrolysate. The pH was adjusted to 5.2 and 6 gm per liter agar (Sigma) added prior to autoclaving. Leaves were cultured under 10 or 40 micromoles per meter squared per second of PAR. In vitro-generated leaves cultured under the higher light intensity did not generate callus. Callus has been subcultured monthly in an effort to establish leaf-free callus cultures. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
There are no outcomes to date.

Publications

• No publications reported this period