Introduction

1. Expansion of a Direct Shoot Organogenesis System in Peanut (Arachishypogaea L.) to Include U.S. Cultivars S. Burns1, M. Gallo1,2, B. Tillman1,3 1Agronomy Department, University of Florida, Gainesville, FL, 2Plant Molecular & Cellular Biology Program, Gainesville, FL, 3North Florida Research & Education Center, Marianna, FL Figure 3. Direct Shoot Organogenesis Trends due to BA Concentration. • Introduction • Currently, the most efficient method for producing transgenic peanut is particle bombardment of somatic embryos. • One major disadvantage of using particle bombardment and somatic embryogenesis is the time (8-12 months) required to produce mature, seed-bearing plants. • Sharma and Anjaiah (2000) developed an alternative system for a Spanish market type, cv. JL-24, that employed direct shoot organogenesis and Agrobacterium-mediated transformation. Mature, transgenic plants were produced as quickly as 4-5 months. Figure 1. Explant Formation & Regeneration of mature A. hypogaea L. (A) Seed morphology and cotyledon explant formation. Arrows indicate the proximal cut end with high regeneration potential. (B) Adventitious shoot buds from cotyledon explants after 3 weeks of culture on SIM. (C) Shoot bud formation on proximal cut end of cotyledon explants after 4 weeks of culture on SIM (2.5X magnification). (D) Shoot development after 2 weeks on shoot elongation medium. (E) Root development after 4 weeks on root induction medium. (E) Mature plant in soil 16 weeks after initial shoot bud formation. 2.5 2 BALevels Vertical Cut (A) Cotyledon A 10 μM Seed Coat 1.5 20 μM 40 μM Mean DSO Rating 80 μM 160 μM Plumule • Hypothesis • The direct shoot organogenesis protocol described by Sharma and Anjaiah (2000) can be expanded and optimized to include readily available U.S. peanut cultivars representing each market type. 1 Cotyledon B Embryo Axis 320 μM 640 μM Radicle W.P. Armstrong 2005 0.5 (B) (C) • Materials & Methods • Peanut cultivars representing the four market types (Virginia, Valencia, Spanish, & Runner) were evaluated for shoot induction from cotyledon explants (Table 1). The protocol used followed that described by Sharma and Anjaiah (2000). • Seed coats were removed and cotyledons were separated into two halves. Cotyledon halves containing the embryo axis were designated “A”, while cotyledons without the embryo axis were designated “B” (Figure 1-A). • Embryo axes were removed from cotyledons A and discarded. Cotyledons A and B were cut in half vertically to obtain quartered-cotyledon explants (Figure 1-A). • The proximal, freshly cut edge of each explant was embedded into shoot induction medium (SIM; MS salts, B5 vitamins, 3% sucrose, 0.8% agar, 10 µM 2,4-Dichlorophenoxyacetic acid, pH 5.8). • Selected cultivars were tested on SIM formulations containing various N6-benzyladenine (BA) levels (Table 1). • Following a four week incubation period, cultures were evaluated on a scale of 1-4 for direct shoot organogenesis (D.S.O.) (Figure 2). • Shoot induction percentages (S.I.%) were determined by using the frequency procedure in SAS. S.I.% represent cultures rating >2 among all evaluated explants for each variety at each BA concentration (Table 2). Mean S.I.% was determined by using the general linear model procedure in SAS with α = 0.05. • Mean D.S.O. ratings were determined using the mixed model procedure in SAS with α = 0.05 (Figure 3). Mean separation was by Tukey-Kramer. 0 GA-G GA-B NM-A VC-2 FLA-07 Error bars:indicateα =0.05 • Results & Conclusions • Cultivars responded differently to the culture conditions. Georgia Green on 40 µM BA had the highest S.I.% (31.2%) and the highest DSO rating (2.22), followed by VC-2 on 10 µM BA (17.3%, 1.84), New Mexico-A on 640 µM BA (15.9%, 1.84), Georgia Brown on 80 µM BA (9.1%, 1.73), and Florida-07 on 40 µM BA (5.6%, 1.82) (Table2, Figure 3). • A difference in shoot induction was observed for each type of cotyledon explant examined. Explant A had a higher S.I.% (15.58 %) and a higher DSO rating (1.75) than explant B (7.7 %, 1.64) (Pr > [t] = 0.0006) (Data not presented in Table 2 or Figure 3). • Cultivars Georgia Green, New Mexico-A and VC-2 appear to be the best suited for future transformation experiments based on their shoot bud production. (F) (D) (E) • Future Work • Optimize tissue culture conditions for other U.S. peanut cultivars. • Quantify expression of a CaMV 35S::uidAexpression-cassettein peanut to develop a highly efficient Agrobacterium transformation protocol (Figure 4). Figure 2. 1-4 Direct Shoot Organogenesis Rating Scheme. (1) Slight greening of explant, no growth; (2) Greening of explant, callus-like growth, no adventitious bud formation; (3) Greening of explant, adventitious bud formation; (4) Greening of explant, adventitious bud formation, small plantlet development (1) (2) (3) (4) Figure 4. Transient Expression of CaMV35S::uidA. Arrows indicate transient GUS-expression on the proximal end of quartered-cotyledon explants of peanut cv. Georgia Green. Explants were inoculated with A. tumefaciens ABI harboring the CaMV 35S::uidAexpression cassette. Table 1. U.S. Peanut Varieties Representing the Four Market Types Tested for Direct Shoot Organogenesis. Table 2. Shoot Induction Percentage of Evaluated Explants. • References • Sharma, KK and V Anjaiah. 2000. An efficient method for the production of transgenic plants of peanut (Arachis hypogaea L.) through Agrobacterium tumefaciens-mediated genetic transformation. Plant Science 159, 7-19. Acknowledgements: Dr. V. James-Hurr, Dr. M. Jain, Dr. Y. Lopez, Mr. J. McKinney, Mr. M. Petefish