Progress on the Sunflower Doubled Haploid Project - National ...

1Xuelin Fu 1 , Richard Horsley 1 ,Lili Qi 2 , Brent Hulke 2 , Chao‐Chein Jan 21NDSU, Dept. of Plant Sciences, Fargo, ND;2USDA‐ARS, NCSL, Fargo, ND

IntroductionAnther cultureOther methods triedFuture plansAcknowledgements2

Doubling the haploid chromosome numberwill produce a doubled haploid (DH).Androgenesis consists of production of plantsfrom anthers or microspores cultured in vitro.Doubled haploids have important applications inplant genetic and breeding research.4

In sunflowers, some methods such as antherculture, microspore culture and irradiation ofpollen grains have been tried by researchersto produce DHs.Anther culture is one of the most usedmethods. Cultivated sunflowers have proven tobe very recalcitrant in anther culture, especiallyfor shoot regeneration.5

Until now, none of the techniques used havebeen successfully applied to Doubled Haploidbreeding programs of sunflowers.In October 2010, the Sunflower DoubledHaploid Project was initiated by the USDA.This report gives the progress of this projectfor 2011.6

Anther cultureBasic processA) inbred lines and amphiploid hybrids were tested.B) Anthers with microspores at the late uninucleatestage were used for culturing, which are light yellowwhitein appearance.microsporesat the lateuninucleatestage7

Embryonic callus or Embryo‐Like‐Structures(ELS) inductionAnther culture of inbred linesHA89, HA410, RHA280, RHA274, Peredovik, RHA801,Seneca and Hopi Dye.Our experiment used L9 (3 4 ) orthogonal design.9

Table 1 L9 (3 4 ) orthogonal design for factors and levels onembryonic callus inductionExperimentandMediumcode1-Genotype2 –Sucrose(g/l)Factors3-Phytohormone(mg/l)4-Organic addition(per litre medium)E1 RHA274 120 BAP0.5+NAA0.5 No additionE2 RHA274 90 BAP1.0+NAA0.5 CH 500mgE3 RHA274 30 BAP0.5+2,4-D 0.5 Coconut water 100mlE4 HA89 120 BAP1.0+NAA0.5 Coconut water 100mlE5 HA89 90 BAP0.5+2,4-D 0.5 No additionE6 HA89 30 BAP0.5+NAA0.5 CH 500mgE7 Peredovik 120 BAP0.5+2,4-D 0.5 CH 500mgE8 Peredovik 90 BAP0.5+NAA0.5 Coconut water 100mlE9 Peredovik 30 BAP1.0+NAA0.5 No addition10

Notes:1) Basic medium: MS (macro +micro‐elements)+Agar 7g/l + Morel and Wetmore’s mediumvitamins + a series of amino acids (AA) .2) BAP‐‐‐6‐benzylaminopurine, 2,4‐D‐‐‐2,4‐dichlorophenoxyacetic acid, NAA‐‐‐naphthlceticacid.3) Every experiment consisted of 3 replications(petri dishes) , each with 150~ 200 anthers.11

Table 4 T Grouping of embryonic callus orELS inductionT Grouping Mean N ExperimentA 205.77 3 E3B 67.87 3 E7B 8.99 3 E2B 5.00 3 E5B 1.78 2 E8B 0.74 2 E9B 0.61 3 E1B 0 3 E6B 0 3 E4Note: Means with the same letter are not significantly different.14

ELS produced in one anthera cluster ofELSdistributedalong thewholeantherELSdevelopmentin anthers ofRHA274 onmedium E3many ELS produced in all fiveanthers of one flower15

In our experiment, we discovered a genotypemediumcombination, E3, which had the highestinduction of ELS.The effectiveness of this medium was confirmedusing more inbred lines and interspecificamphiploids.16

Table 5 Induction results of embryonic callus or ELSMedium Material No. of anthersculturedNo. ofcallus orELSrate of callus(%)E3 HA89 350 10 2.86±0.12E3 HA410 575 109 18.96±1.43E3 RHA274 530 909 171.51±20.67E3 Peredovik 370 50 13.51±18.61E3 RHA801 755 427 56.56±3.58E3 Seneca 360 92 25.56±3.14Amount ofcallusinductionchangedamonggenotypes,butimprovedon thewhole.E3 Hopi Dye 315 175 55.55±0.30E3 G08/2260 200 48 24.00±40.96E3 G08/2263 470 95 20.21±6.79E3 G08/2266 305 27 8.85±3.1417

HA410‐‐‐anther baseswelledthencallusedRHA280‐‐‐antherwallcallusedResponses of anthers of different genotypes oninduction medium18

Anther culture of amphiploid hybrids% of callus inductionMaterialFigure 1 Anther callus induction of amphiploid hybrids1‐A: anthers with microspores at the late uninucleate stage4‐A: anthers with microspores at or after the binucleate stage19

Effects of chemical inducers on anther cultureThree chemical inducers were tested on anther cultures:2‐hydroxynicotinic acid (2‐HNA),2‐(p‐chlorophenoxy)‐2‐methylpropionic acid (PCIB),24‐epibrassinolide (EBR).Eight inbred lines were used : HA89, HA410, RHA280,RHA274, Peredovik, RHA801,Seneca, and Hopi Dye.20

For chemical inducer 2‐HNA, sunflower heads with10‐cm long stems were precultured in 2‐HNAsolutions at 32 o C in the dark. Four combinations of2‐HNA concentration and time were used.Pretreatment of sunflower heads by 2‐HNA2‐HNA 100mg/l 50mg/lPretreatmentTime Code48 h a b72 h e f0 CK21

Table 6 Anther callus induction results by 2‐HNA pretreatmentMaterialAndNo. ofanthersNo. ofcallusRate ofcallusMaterialAndNo. ofanthersNo. ofcallusRate ofcalluspretreatment cultured(%) pretreatment cultured(%)codecodeHA89‐a 495 12 2.42 Peredovik‐a 725 56 7.72HA89‐b 430 14 3.26 Peredovik‐b 540 83 15.37HA89‐e 565 14 2.48 Peredovik‐e 380 3 0.79HA89‐f 290 1 0.34 Peredovik‐f 250 1 0.45HA89‐CK 728 113 15.52 Peredovik‐CK 652 62 9.51HA410‐a 360 20 5.56 RHA801‐a 545 27 4.95HA410‐b 340 22 6.47 RHA801‐b 195 1 0.51HA410‐e 390 2 3.08 RHA801‐e 810 14 1.73HA410‐f 425 18 4.24 RHA801‐f 365 0 0HA410‐CK 419 16 3.82 RHA801‐CK 165 25 15.15Callus induction didRHA280‐a 170 3 1.76 Seneca‐a 410 10 2.44RHA280‐b 190 23 12.11 Seneca‐b not 585 increase. 3 0.51RHA280‐e 470 32 6.81 Seneca‐e 200 7 3.5RHA280‐f 0 0 0 Seneca‐f 410 4 0.98RHA280‐CK 630 8 1.26 Seneca‐CK 1300 183 14.08RHA274‐a 395 51 12.91 Hopi Dye‐a 130 8 6.15RHA274‐b 325 64 19.69 Hopi Dye‐b 350 4 1.14RHA274‐e 615 55 8.94 Hopi Dye‐e 595 30 5.04RHA274‐f 660 0 0 Hopi Dye‐f 380 48 12.63RHA274‐CK 1130 138 12.21 Hopi Dye‐CK 660 126 19.0922

Table 7 Anther callus induction results by adding PCIB and EBRMaterialandMediumNo. ofantherculturedNo. ofcallusRate ofcallus(%)HA89‐A1 103 0 0HA89‐A3 104 1 0.96MaterialandMediumNo. ofantherculturedNo. ofcallusRate ofcallus(%)Peredovik ‐A1 128 3 2.34Peredovik ‐A3 116 1 0.86HA410‐A1 112 0 0.00 RHA801‐A1 48 0 0.00HA410‐A3 115 0 0.00 RHA801‐A3 53 0 0.00RHA280‐PCIB nor 0.1 µMA1 46 0 0.00 Seneca ‐A1 96 4 4.17RHA280‐effect onA3 58 0 0.00 Seneca ‐A3 130 1 0.77improvingRHA274‐Hopi Dye ‐induction of antherA1 88 1 1.14A1 92 3 3.26RHA274‐A3 81 1 1.23Neither 10 µMEBR had a positivecalli.Hopi Dye ‐A3 119 0 0.00Note: A1‐‐‐ PCIB 10µM; A3‐‐‐ EBR 0.1µM.23

Plant regeneration of embryonic callus and ELSMedia codeS1S2S3S4S5S6S7S8S9S10Plant regeneration mediaIngredientsMS + BAP 0.5mg/l + NAA0.5mg/l+ Sucrose 20g/l +Agar 7g/lMS + BAP 0.5mg/l + NAA0.5mg/l+ Sucrose 20g/l +Agar 7g/l+ coconutwater 100ml/lMS + BAP 1.0 mg/l + NAA0.5mg/l+ Sucrose 20g/l +Agar 7g/lMS + BAP 1.0 mg/l + NAA0.5mg/l+ Sucrose 20g/l +Agar 7g/l+coconut water 100ml/lMS + BAP 0.5mg/l+ Sucrose 20g/l +Agar 7g/l+ coconut liquid 100ml/lMS + BAP 0.25mg/l + Sucrose 20g/l +Agar 7g/lMS + Sucrose 20g/l +Agar 7g/lMS + BAP 0.5 mg/l + NAA0.1mg/l+ Sucrose 20g/l +Agar 7g/lMS + BAP 0.5mg/l + IBA0.1mg/l+ Sucrose 30g/l +Agar 7g/lMS + BAP 0.5 mg/l + NAA0.1mg/l+ Sucrose 20g/l +Agar 7g/l+coconut water 100ml/lIBA‐‐‐Indole‐3‐butyric acid.24

% of green spots andsurvival calliMaterialand mediaM4‐‐‐RHA274, M5‐‐‐PeredovikFigure 2 The first sublture of anther callus: regeneration results of twoinbred lines25

Medium S2 produced a higher green spotconversion rate (about 35%) than the othersduring the first subculture for plant regeneration.Many subculture steps are normally required toproduce shoots from callus or ELS.During the subculture, some genotypes such asRHA274 and amphiploids of H. pumilus ×P21,produced embryoids from callus or ELS throughsecondary embryogenesis, then proliferated.26

1 stsubculture ofELS on S2medium2 nd subculture:secondaryembryogenesisoccuredPlantregenerationprocess ofRHA2743 rdsubculture:embryoidsproliferated27

EmbryoidgerminationEmbryoid on regenerationmediumSmall shootelongationEmbryoid at differentdevelopmental stagesPlant regenerationprocess of amphiploidH. pumilus ×P21Normal plantlets Abnormal plantlet28

For plant regeneration, the inbred lines did notproduce shoots from callus or ELS, unlike theamphiploids.Amphiploid H. pumilus ×P21 had the most plantregeneration of the experimental materials used.More than 100 plantlets have been produced inrooting media and soils to date.29

NMS HA89×H. maximiliani,amphiploidH. pumilus×P21,amphiploidShoot elongationon hormone‐freemedium beforetransferring torooting medium30

Rooting of the regenerated plantletsMediacoder1r2r3r4r1ar2ar3ar4aRooting mediaIngredients½ MS + NAA1.0mg/l+ Sucrose 20g/l +Agar 7g/l½MS + IBA 1.0mg/l+ Sucrose 20g/l +Agar 7g/l½ MS + NAA0.5mg/l+ Sucrose 20g/l +Agar 7g/l½MS + IBA 0.5 mg/l+ Sucrose 20g/l +Agar 7g/l½ MS + NAA1.0mg/l+ Sucrose 20g/l +Agar 7g/l+ AC 0.5g/l½MS + IBA 1.0mg/l+ Sucrose 20g/l +Agar 7g/l+ AC 0.5g/l½ MS + NAA0.5mg/l+ Sucrose 20g/l +Agar 7g/l+ AC 0.5g/l½MS + IBA 0.5 mg/l+ Sucrose 20g/l +Agar 7g/l+ AC 0.5g/lAC‐‐‐‐activated charcoal31

Rooting rate %MediaFigure 3 Rooting rate of amphiploid H. pumilus ×P21 shootsr2a medium has the highest rooting rate among all theexperimental media. Adding Activated Charcoal to therooting medium is helpful for shoot rooting.32

+ AC ‐ACShoots in rooting mediumPlantletstransplanted topeat pellets33

Our results produced plantlets from anther culture ofthree amphiploid hybrids, NMS HA89 ×H. maximiliani ,H. pumilus ×P21, and (H.Hirsutus×P21) × HA89.Some of the plantlets rooted successfully.Chromosome numbers of these plantlets will bedetermined.34

Foreign pollen inducersInterspecific hybridizations between cultivatedsunflowers and H. tuberosus were made. Twenty onehybrid seeds were obtained.Female parent(♀)Male parent(♂)No. ofheadspollinatedNo. of hybridseedsharvestedPercentseed set(%)(CMS HA412HO×HA467) F 1(CMS HA412HO×HA467) F 1(CMS HA412HO×HA467) F 1H. tuberosus(West Fargo)H. tuberosus(KindredSouth)H. tuberosus(Moorhead)4 10 0.3572 10 0.7143 1 0.04835

GreenhypocotylRedhypocotylRedhypocotylThe hybrid seeds are germinating in culture.These three seedlings all derived from the combination:(CMS HA412HO ×HA467) F 1× H. tuberosus (Kindred South).36

Future plans• Transition from anther culture to microsporeculture.• Continue “Foreign pollen inducer” work. Detecthaploids by counting chromosome of the hybridseedlings.•Pursue “Induced mutation” work. Dr. Brent Hulkeplans to testcross M2 progenies to determinewhether or not any individual mutation line leadsto haploid progeny.• Establish true haploid lines, develop doubledhaploid lines through chromosome doubling.37

AcknowledgementsLisa BrownLeonard CookBrady VickJayma Moore (OCD‐EM Lab)Zahirul TalukderZhao LiuAlicia GarciaAngelia HognessMarjorie OlsonNational Sunflower Association38