Introduction
1
Introduction Introduction Currently, the most efficient method for Currently, the most efficient method for producing transgenic peanut is particle producing transgenic peanut is particle bombardment of somatic embryos. bombardment of somatic embryos. One major disadvantage of using particle One major disadvantage of using particle bombardment and somatic embryogenesis is the bombardment and somatic embryogenesis is the time (8-12 months) required to produce mature, time (8-12 months) required to produce mature, seed-bearing plants. seed-bearing plants. Sharma and Anjaiah (2000) developed an Sharma and Anjaiah (2000) developed an alternative system for a Spanish market type, alternative system for a Spanish market type, cv. JL-24, that employed direct shoot cv. JL-24, that employed direct shoot organogenesis and organogenesis and Agrobacterium Agrobacterium -mediated -mediated transformation. Mature, transgenic plants were transformation. Mature, transgenic plants were produced as quickly as 4-5 months. produced as quickly as 4-5 months. Hypothesis Hypothesis The direct shoot organogenesis protocol The direct shoot organogenesis protocol described by Sharma and Anjaiah (2000) can be described by Sharma and Anjaiah (2000) can be expanded and optimized to include readily expanded and optimized to include readily available U.S. peanut cultivars representing each available U.S. peanut cultivars representing each market type. market type. Materials & Methods Materials & Methods Peanut cultivars representing the four market Peanut cultivars representing the four market types (Virginia, Valencia, Spanish, & Runner) types (Virginia, Valencia, Spanish, & Runner) were evaluated for shoot induction from cotyledon were evaluated for shoot induction from cotyledon explants (Table 1). The protocol used followed explants (Table 1). The protocol used followed that described by Sharma and Anjaiah (2000). that described by Sharma and Anjaiah (2000). Seed coats were removed and cotyledons were Seed coats were removed and cotyledons were separated into two halves. Cotyledon halves separated into two halves. Cotyledon halves containing the embryo axis were designated “A”, containing the embryo axis were designated “A”, while cotyledons without the embryo axis were while cotyledons without the embryo axis were designated “B” (Figure 1-A). designated “B” (Figure 1-A). Embryo axes were removed from cotyledons A and Embryo axes were removed from cotyledons A and discarded. Cotyledons A and B were cut in half discarded. Cotyledons A and B were cut in half vertically to obtain quartered-cotyledon explants vertically to obtain quartered-cotyledon explants (Figure 1-A). (Figure 1-A). The proximal, freshly cut edge of each explant The proximal, freshly cut edge of each explant was embedded into shoot induction medium (SIM; MS was embedded into shoot induction medium (SIM; MS salts, B5 vitamins, 3% sucrose, 0.8% agar, 10 µM salts, B5 vitamins, 3% sucrose, 0.8% agar, 10 µM 2,4-Dichlorophenoxyacetic acid, pH 5.8). 2,4-Dichlorophenoxyacetic acid, pH 5.8). Selected cultivars were tested on SIM Selected cultivars were tested on SIM formulations containing various N formulations containing various N 6 6 -benzyladenine -benzyladenine (BA) levels (Table 1). (BA) levels (Table 1). Following a four week incubation period, Following a four week incubation period, cultures were evaluated on a scale of 1-4 for cultures were evaluated on a scale of 1-4 for direct shoot organogenesis (D.S.O.) (Figure 2). direct shoot organogenesis (D.S.O.) (Figure 2). Shoot induction percentages (S.I.%) were Shoot induction percentages (S.I.%) were determined by using the frequency procedure in determined by using the frequency procedure in SAS. S.I.% represent cultures rating >2 among all SAS. S.I.% represent cultures rating >2 among all evaluated explants for each variety at each BA evaluated explants for each variety at each BA concentration (Table 2). Mean S.I.% was concentration (Table 2). Mean S.I.% was determined by using the general linear model determined by using the general linear model procedure in SAS with α = 0.05. procedure in SAS with α = 0.05. Mean D.S.O. ratings were determined using the Mean D.S.O. ratings were determined using the mixed model procedure in SAS with α = 0.05 mixed model procedure in SAS with α = 0.05 (Figure 3). Mean separation was by Tukey-Kramer. (Figure 3). Mean separation was by Tukey-Kramer. Results & Conclusions Results & Conclusions Cultivars responded differently to the culture Cultivars responded differently to the culture conditions. Georgia Green on 40 µM BA had the conditions. Georgia Green on 40 µM BA had the highest S.I.% (31.2%) and the highest DSO rating highest S.I.% (31.2%) and the highest DSO rating (2.22), followed by VC-2 on 10 µM BA (17.3%, (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), 1.84), New Mexico-A on 640 µM BA (15.9%, 1.84), Georgia Brown on 80 µM BA (9.1%, 1.73), and Georgia Brown on 80 µM BA (9.1%, 1.73), and Florida-07 on 40 µM BA (5.6%, 1.82) (Table2, Florida-07 on 40 µM BA (5.6%, 1.82) (Table2, Figure 3). Figure 3). A difference in shoot induction was observed A difference in shoot induction was observed for each type of cotyledon explant examined. for each type of cotyledon explant examined. Explant A had a higher S.I.% (15.58 %) and a Explant A had a higher S.I.% (15.58 %) and a higher DSO rating (1.75) than explant B (7.7 %, higher DSO rating (1.75) than explant B (7.7 %, 1.64) (Pr > [t] = 0.0006) (Data not presented in 1.64) (Pr > [t] = 0.0006) (Data not presented in Table 2 or Figure 3). Table 2 or Figure 3). Cultivars Georgia Green, New Mexico-A and VC-2 Cultivars Georgia Green, New Mexico-A and VC-2 appear to be the best suited for future appear to be the best suited for future transformation experiments based on their shoot transformation experiments based on their shoot bud production. bud production. Future Work Future Work Optimize tissue culture conditions for other Optimize tissue culture conditions for other U.S. peanut cultivars. U.S. peanut cultivars. Quantify expression of a CaMV 35S:: Quantify expression of a CaMV 35S:: uidA uidA expression-cassette expression-cassette in peanut to develop a highly in peanut to develop a highly efficient efficient Agrobacterium Agrobacterium transformation protocol transformation protocol (Figure 4). (Figure 4). References References Sharma, KK and V Anjaiah. 2000. An efficient method for Sharma, KK and V Anjaiah. 2000. An efficient method for the production of transgenic plants of peanut ( the production of transgenic plants of peanut ( Arachis Arachis hypogaea hypogaea L.) through L.) through Agrobacterium tumefaciens Agrobacterium tumefaciens -mediated -mediated genetic transformation. Plant Science 159, 7-19. genetic transformation. Plant Science 159, 7-19. Acknowledgements Acknowledgements : Dr. V. James-Hurr, Dr. M. Jain, Dr. Y. Lopez, Mr. J. : Dr. V. James-Hurr, Dr. M. Jain, Dr. Y. Lopez, Mr. J. McKinney, Mr. M. Petefish McKinney, Mr. M. Petefish Expansion of a Direct Shoot Organogenesis System in Peanut ( Expansion of a Direct Shoot Organogenesis System in Peanut ( Arachis hypogaea Arachis hypogaea L.) L.) to Include U.S. Cultivars to Include U.S. Cultivars S. Burns S. Burns 1 1 , M. Gallo , M. Gallo 1,2 1,2 , B. Tillman , B. Tillman 1,3 1,3 1 1 Agronomy Department, University of Florida, Gainesville, FL, Agronomy Department, University of Florida, Gainesville, FL, 2 2 Plant Molecular & Cellular Biology Program, Gainesville, FL, Plant Molecular & Cellular Biology Program, Gainesville, FL, 3 3 North Florida Research & Education Center, North Florida Research & Education Center, Marianna, FL Marianna, FL Cultivar Cultivar Market Type Market Type BA levels tested (μM) BA levels tested (μM) Florida-07 Florida-07 Runner Runner 10, 20, 40, 80 10, 20, 40, 80 Georgia Green Georgia Green Runner Runner 10, 20, 40, 80 10, 20, 40, 80 Georgia Browne Georgia Browne Spanish Spanish 10, 20, 40, 80, 160, 320 10, 20, 40, 80, 160, 320 New Mexico-A New Mexico-A Valencia Valencia 10, 20, 40, 80, 160, 10, 20, 40, 80, 160, 320, 640 320, 640 VC-2 VC-2 Virginia Virginia 10, 20, 40, 80 10, 20, 40, 80 FLA-07 FLA-07 GA-G GA-G GA-B GA-B NM-A NM-A VC-2 VC-2 10 10 μ μ M M 20 20 μ μ M M 40 40 μ μ M M 80 80 μ μ M M 160 160 μ μ M M 320 320 μ μ M M 640 640 μ μ M M 0 0 0.5 0.5 1.5 1.5 2 2 2.5 2.5 1 1 Mean DSO Rating Mean DSO Rating Cotyledon Cotyledon A A Cotyledon B Cotyledon B Seed Coat Seed Coat Plumule Plumule Radicle Radicle Embry Embry o o Axis Axis Vertical Cut Vertical Cut BA BA Levels Levels Figure 1. Explant Formation & Regeneration of Figure 1. Explant Formation & Regeneration of mature mature A. hypogaea A. hypogaea L. L. (A) Seed morphology and cotyledon explant formation. Arrows (A) Seed morphology and cotyledon explant formation. Arrows indicate the proximal cut end with high regeneration potential. indicate the proximal cut end with high regeneration potential. (B) Adventitious shoot buds from cotyledon explants after 3 (B) Adventitious shoot buds from cotyledon explants after 3 weeks of culture on SIM. (C) Shoot bud formation on proximal weeks of culture on SIM. (C) Shoot bud formation on proximal cut end of cotyledon explants after 4 weeks of culture on SIM cut end of cotyledon explants after 4 weeks of culture on SIM (2.5X magnification). (D) Shoot development after 2 weeks on (2.5X magnification). (D) Shoot development after 2 weeks on shoot elongation medium. (E) Root development after 4 weeks on shoot elongation medium. (E) Root development after 4 weeks on root induction medium. (E) Mature plant in soil 16 weeks after root induction medium. (E) Mature plant in soil 16 weeks after initial shoot bud formation. initial shoot bud formation. (1) Slight greening of explant, no growth; (2) Greening of explant, (1) Slight greening of explant, no growth; (2) Greening of explant, callus-like growth, no adventitious bud formation; (3) Greening of callus-like growth, no adventitious bud formation; (3) Greening of explant, adventitious bud formation; (4) Greening of explant, explant, adventitious bud formation; (4) Greening of explant, adventitious bud formation, small plantlet development adventitious bud formation, small plantlet development Arrows indicate transient GUS-expression on the proximal end of Arrows indicate transient GUS-expression on the proximal end of quartered-cotyledon explants of peanut cv. Georgia Green. quartered-cotyledon explants of peanut cv. Georgia Green. Explants were inoculated with Explants were inoculated with A. tumefaciens A. tumefaciens ABI harboring the ABI harboring the CaMV 35S:: CaMV 35S:: uidA uidA expression cassette. expression cassette. Table 1. U.S. Peanut Varieties Representing the Table 1. U.S. Peanut Varieties Representing the Four Market Types Tested for Direct Shoot Four Market Types Tested for Direct Shoot Organogenesis. Organogenesis. Figure 3. Direct Shoot Organogenesis Trends due to Figure 3. Direct Shoot Organogenesis Trends due to BA Concentration. BA Concentration. (1 (1 ) ) (2 (2 ) ) (3 (3 ) ) (4 (4 ) ) (A (A ) ) (B (B ) ) (C (C ) ) (D (D ) ) (E (E ) ) (F (F ) ) W.P. Armstrong 2005 W.P. Armstrong 2005 Table 2. Shoot Induction Percentage of Evaluated Table 2. Shoot Induction Percentage of Evaluated Explants. Explants. Figure 2. 1-4 Direct Shoot Organogenesis Figure 2. 1-4 Direct Shoot Organogenesis Rating Scheme. Rating Scheme. Figure 4. Transient Expression of CaMV35S:: Figure 4. Transient Expression of CaMV35S:: uidA uidA . . Error bars:indicate Error bars:indicate α α =0.05 =0.05
description
Expansion of a Direct Shoot Organogenesis System in Peanut ( Arachis hypogaea L.) to Include U.S. Cultivars S. Burns 1 , M. Gallo 1,2 , B. Tillman 1,3 - PowerPoint PPT Presentation
Transcript of Introduction
IntroductionIntroductionCurrently, the most efficient method for producing transgenic Currently, the most efficient method for producing transgenic peanut is particle bombardment of somatic embryos. peanut is particle bombardment of somatic embryos.
One major disadvantage of using particle bombardment and One major disadvantage of using particle bombardment and somatic embryogenesis is the time (8-12 months) required to somatic embryogenesis is the time (8-12 months) required to produce mature, seed-bearing plants.produce mature, seed-bearing plants.
Sharma and Anjaiah (2000) developed an alternative system for a Sharma and Anjaiah (2000) developed an alternative system for a Spanish market type, cv. JL-24, that employed direct shoot Spanish market type, cv. JL-24, that employed direct shoot organogenesis and organogenesis and AgrobacteriumAgrobacterium-mediated transformation. -mediated transformation. Mature, transgenic plants were produced as quickly as 4-5 months.Mature, transgenic plants were produced as quickly as 4-5 months.
HypothesisHypothesisThe direct shoot organogenesis protocol described by Sharma and The direct shoot organogenesis protocol described by Sharma and Anjaiah (2000) can be expanded and optimized to include readily Anjaiah (2000) can be expanded and optimized to include readily available U.S. peanut cultivars representing each market type. available U.S. peanut cultivars representing each market type.
Materials & MethodsMaterials & MethodsPeanut cultivars representing the four market types (Virginia, Peanut cultivars representing the four market types (Virginia, Valencia, Spanish, & Runner) were evaluated for shoot induction Valencia, Spanish, & Runner) were evaluated for shoot induction from cotyledon explants (Table 1). The protocol used followed that from cotyledon explants (Table 1). The protocol used followed that described by Sharma and Anjaiah (2000).described by Sharma and Anjaiah (2000).
Seed coats were removed and cotyledons were separated into two Seed coats were removed and cotyledons were separated into two halves. Cotyledon halves containing the embryo axis were designated halves. Cotyledon halves containing the embryo axis were designated “A”, while cotyledons without the embryo axis were designated “B” “A”, while cotyledons without the embryo axis were designated “B” (Figure 1-A). (Figure 1-A).
Embryo axes were removed from cotyledons A and discarded. Embryo axes were removed from cotyledons A and discarded. Cotyledons A and B were cut in half vertically to obtain quartered-Cotyledons A and B were cut in half vertically to obtain quartered-cotyledon explants (Figure 1-A). cotyledon explants (Figure 1-A).
The proximal, freshly cut edge of each explant was embedded into The proximal, freshly cut edge of each explant was embedded into shoot induction medium (SIM; MS salts, B5 vitamins, 3% sucrose, shoot induction medium (SIM; MS salts, B5 vitamins, 3% sucrose, 0.8% agar, 10 µM 2,4-Dichlorophenoxyacetic acid, pH 5.8).0.8% agar, 10 µM 2,4-Dichlorophenoxyacetic acid, pH 5.8).
Selected cultivars were tested on SIM formulations containing Selected cultivars were tested on SIM formulations containing various Nvarious N66-benzyladenine (BA) levels (Table 1). -benzyladenine (BA) levels (Table 1).
Following a four week incubation period, cultures were evaluated Following a four week incubation period, cultures were evaluated on a scale of 1-4 for direct shoot organogenesis (D.S.O.) (Figure 2). 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 Shoot induction percentages (S.I.%) were determined by using the frequency procedure in SAS. S.I.% represent cultures rating >2 frequency procedure in SAS. S.I.% represent cultures rating >2 among all evaluated explants for each variety at each BA among all evaluated explants for each variety at each BA concentration (Table 2). Mean S.I.% was determined by using the concentration (Table 2). Mean S.I.% was determined by using the general linear model procedure in SAS with α = 0.05. general linear model procedure in SAS with α = 0.05.
Mean D.S.O. ratings were determined using the mixed model Mean D.S.O. ratings were determined using the mixed model procedure in SAS with α = 0.05 (Figure 3). Mean separation was by procedure in SAS with α = 0.05 (Figure 3). Mean separation was by Tukey-Kramer. Tukey-Kramer.
Results & ConclusionsResults & ConclusionsCultivars responded differently to the culture conditions. Georgia Cultivars responded differently to the culture conditions. Georgia Green on 40 µM BA had the highest S.I.% (31.2%) and the highest 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), 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 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) µM BA (9.1%, 1.73), and Florida-07 on 40 µM BA (5.6%, 1.82) (Table2, Figure 3).(Table2, Figure 3).
A difference in shoot induction was observed for each type of A difference in shoot induction was observed for each type of cotyledon explant examined. Explant A had a higher S.I.% (15.58 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 %) 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).> [t] = 0.0006) (Data not presented in Table 2 or Figure 3).
Cultivars Georgia Green, New Mexico-A and VC-2 appear to be Cultivars Georgia Green, New Mexico-A and VC-2 appear to be the best suited for future transformation experiments based on their the best suited for future transformation experiments based on their shoot bud production. shoot bud production. Future WorkFuture WorkOptimize tissue culture conditions for other U.S. peanut cultivars.Optimize tissue culture conditions for other U.S. peanut cultivars.
Quantify expression of a CaMV 35S::Quantify expression of a CaMV 35S::uidA uidA expression-cassetteexpression-cassette in in peanut to develop a highly efficient peanut to develop a highly efficient AgrobacteriumAgrobacterium transformation transformation protocol (Figure 4). protocol (Figure 4).
ReferencesReferencesSharma, KK and V Anjaiah. 2000. An efficient method for the production of Sharma, KK and V Anjaiah. 2000. An efficient method for the production of
transgenic plants of peanut (transgenic plants of peanut (Arachis hypogaeaArachis hypogaea L.) through L.) through Agrobacterium Agrobacterium tumefacienstumefaciens-mediated genetic transformation. Plant Science 159, 7-19.-mediated genetic transformation. Plant Science 159, 7-19.
AcknowledgementsAcknowledgements: Dr. V. James-Hurr, Dr. M. Jain, Dr. Y. Lopez, Mr. J. McKinney, Mr. M. : Dr. V. James-Hurr, Dr. M. Jain, Dr. Y. Lopez, Mr. J. McKinney, Mr. M. Petefish Petefish
Expansion of a Direct Shoot Organogenesis System in Peanut (Expansion of a Direct Shoot Organogenesis System in Peanut (Arachis hypogaeaArachis hypogaea L.) to Include U.S. Cultivars L.) to Include U.S. CultivarsS. BurnsS. Burns11, M. Gallo, M. Gallo1,21,2, B. Tillman, B. Tillman1,31,3
11Agronomy Department, University of Florida, Gainesville, FL, Agronomy Department, University of Florida, Gainesville, FL, 22Plant Molecular & Cellular Biology Program, Gainesville, FL, Plant Molecular & Cellular Biology Program, Gainesville, FL, 33North Florida Research & Education Center, Marianna, FL North Florida Research & Education Center, Marianna, FL
CultivarCultivar Market TypeMarket Type BA levels tested (μM)BA levels tested (μM)
Florida-07Florida-07 RunnerRunner 10, 20, 40, 8010, 20, 40, 80
Georgia GreenGeorgia Green RunnerRunner 10, 20, 40, 8010, 20, 40, 80
Georgia BrowneGeorgia Browne SpanishSpanish 10, 20, 40, 80, 160, 32010, 20, 40, 80, 160, 320
New Mexico-ANew Mexico-A ValenciaValencia 10, 20, 40, 80, 160, 320, 64010, 20, 40, 80, 160, 320, 640
VC-2VC-2 VirginiaVirginia 10, 20, 40, 8010, 20, 40, 80
FLA-07FLA-07 GA-GGA-G GA-BGA-B NM-ANM-A VC-2VC-2
10 10 μμMM
20 20 μμMM
40 40 μμMM
80 80 μμMM
160 160 μμMM
320 320 μμMM
640 640 μμMM
00
0.50.5
1.51.5
22
2.52.5
11
Mea
n D
SO R
atin
gM
ean
DSO
Rat
ingCotyledon ACotyledon A
Cotyledon BCotyledon B
Seed CoatSeed Coat
PlumulePlumule
RadicleRadicleEmbryo Embryo
AxisAxis
Vertical CutVertical Cut BABA LevelsLevels
Figure 1. Explant Formation & Regeneration of mature Figure 1. Explant Formation & Regeneration of mature A. hypogaeaA. hypogaea L. L.(A) Seed morphology and cotyledon explant formation. Arrows indicate the proximal cut (A) Seed morphology and cotyledon explant formation. Arrows indicate the proximal cut end with high regeneration potential. (B) Adventitious shoot buds from cotyledon 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 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 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 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 weeks on root induction medium. (E) Mature plant in soil 16 weeks after initial shoot bud formation.formation.
(1) Slight greening of explant, no growth; (2) Greening of explant, callus-like growth, no adventitious (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, bud formation; (3) Greening of explant, adventitious bud formation; (4) Greening of explant, adventitious bud formation, small plantlet developmentadventitious bud formation, small plantlet development
Arrows indicate transient GUS-expression on the proximal end of quartered-cotyledon Arrows indicate transient GUS-expression on the proximal end of quartered-cotyledon explants of peanut cv. Georgia Green. Explants were inoculated with explants of peanut cv. Georgia Green. Explants were inoculated with A. tumefaciensA. tumefaciens ABI ABI harboring the CaMV 35S::harboring the CaMV 35S::uidA uidA expression cassette.expression cassette.
Table 1. U.S. Peanut Varieties Representing the Four Market Types Table 1. U.S. Peanut Varieties Representing the Four Market Types Tested for Direct Shoot Organogenesis.Tested for Direct Shoot Organogenesis.
Figure 3. Direct Shoot Organogenesis Trends due to BA Concentration. Figure 3. Direct Shoot Organogenesis Trends due to BA Concentration.
(1)(1) (2)(2) (3)(3) (4)(4)
(A)(A)
(B)(B) (C)(C)
(D)(D) (E)(E) (F)(F)
W.P. Armstrong 2005W.P. Armstrong 2005
Table 2. Shoot Induction Percentage of Evaluated Explants.Table 2. Shoot Induction Percentage of Evaluated Explants.
Figure 2. 1-4 Direct Shoot Organogenesis Rating Scheme.Figure 2. 1-4 Direct Shoot Organogenesis Rating Scheme.
Figure 4. Transient Expression of CaMV35S::Figure 4. Transient Expression of CaMV35S::uidAuidA..
Error bars:indicate Error bars:indicate αα =0.05 =0.05
