anther culture potential of indica rice varieties, kurulu thuda and bg ...

Tropical Agricultural Research & Extension 12(2): 2009ANTHER CULTURE POTENTIAL OF INDICA RICE VARIETIES, KURULU THUDAAND BG 250TD Silva* and WJ RatnayakeDepartment of Plant Sciences, University of Colombo, Sri LankaAccepted: 7 th October 2009ABSTRACTThe anther culture ability of two local indica rice varieties were evaluated by anther plating on N6 andSK-I callus induction media. The traditional rice variety, Kurulu Thuda displayed good anther culturepotential with a callus induction frequency of 17.2%. Anthers most suitable for culture were those bearingbi-nucleate pollen. Anther response was relatively poor in the new improved variety, Bg 250 (1.4%).Callus induction was nearly three times higher on N6 medium than on SK-I medium in var. KuruluThuda. Shoot regeneration was achieved in var. Kurulu Thuda on SK-II medium, but only from callusinduced on N6 medium. From a total of 115 calli transferred from N6 to regeneration medium shootdevelopment occurred in 7 calli, thus limiting the overall anther culture efficiency. Of the calli withmorphogenetic competence, 5 gave rise to albino shoots and 2 produced green shoots. After root developmentthe green plantlets were potted in soil. Plant regeneration could not be observed from callusinduced in var. Bg 250.Key words: Anther culture, Callus induction, Indica riceINTRODUCTIONAnther culture is an innovative technique that canbe utilized for the rapid development of homozygousplants. Pollen within the cultured anthers maybe induced to form callus which will later regeneratehaploid plants. Haploids upon chromosomedoubling yield fully homozygous genotypes. Thetechnique has been used successfully to producehomozygous breeding lines in japonica rice (Brarand Kush 2006). However, the potential for indicarice anther culture is yet to be fully exploited due tovarious constraints that include a recalcitrant geneticbackground in the indica varieties (He et al.2006). Several attempts at anther culture of localindica rice germplasm have also met with limitedsuccess (Kumari et al. 2006; Silva and Achala2008; Rathieka and Silva 2007).The ability of pollen to redirect its developmentpathway from gametogenesis to embryogenesisdepends on the stage of maturity of the pollengrains at the time of culture. For rice, the best stagehas been described as uni-nucleate to early bi nucleatestage (Jahne and Lorz 1995). Therefore, determiningthe development stage of pollen in theanthers is important to optimize the anther cultureresponse of a given rice genotype (Silva 2010). Improvedstaining procedures have been used to identifyrice pollen nuclei (Gupta and Borthakur 1987).A visual marker that shows good correlation withthe pollen stage is used as a guide to identify therequired stage of pollen during large scale culturing.Usually, the distance between the collar of theflag leaf and the ligule of the penultimate leaf of thetiller serves as a reliable guide to anther maturity(Bishnoi et al. 2000). The objective of the experimentwas to identify the anther culture potential ofthe rice varieties, Kurulu Thuda and Bg 250, facilitatedby defining the appropriate stage of pollendevelopment for successful anther culture.MATERIALS AND METHODSPollen staging using iron alum haematoxylinstainThe stage of pollen division was studied in anthersharvested at different maturity stages in KuruluThuda using iron-alum haematoxylin staining procedure(Gupta and Borthakur 1987). Panicle maturitywas correlated with the distance between theflag leaf and the penultimate leaf collar. Paniclesenclosed in leaf sheaths were harvested when thisdistance was 3, 4, 5, 6, 7 and 8cm. From each harvestedpanicle, spikelets were removed from theupper, mid, and lower portion that represented amaturity gradient of the florets. Three spikeletswere sampled per region of the panicle, and fromeach spikelet a single anther was selected randomlyfor staining. The dissected out anther was dipped in4% iron alum haematoxylin stain in a coveredwatch glass. The time required for differentialstaining of the nuclei was worked out for pollengrains of different maturity by trial and error. Accordingly,very young pollen collected from panicleswhen the distance between the flag leaf and thepenultimate leaf auricle was 3cm were stained forup to 5min. and the duration of staining was in-*Corresponding authorPaper presented at the 2nd National Symposium, Faculty of Agriculture, University of Ruhuna

52TD SILVA AND WJ RATNAYAKE : ANTHER CULTURE OF INDICA RICEcreased up to 25, 60, 110, 130 and 140min. whenthis distance was 4, 5, 6, 7 and 8cm respectively.Stained anthers were squashed on a clean glassslide with a drop of haematoxylin stain, and observedunder the light microscope for stages of pollendevelopment.Anther CultureAnthers were dissected out from panicles harvestedat similar maturity stages as those used for pollenstaging. The panicles enclosed in leaf sheaths werewiped with 70% alcohol, and wrapped in aluminumfoil. Foil-wrapped and labeled panicles were placedin sealed plastic bags and subjected to cold pretreatmentat 10 0 C for 7d. Following cold-shock,panicles were surface sterilized in 20% Clorox for20min. Anthers were dissected out in the laminarflow bench from separated spikelets. Each spikeletwas snipped at the base while holding from the tip,to detach the anther lobes from the filaments. Thereleased anthers were dropped onto agar solidifiedmedia contained in Petri dishes. Anthers were madeto spread evenly on the agar surface by rotating thePetri dish during anther-plating. Anthers of the twovarieties were cultured on N6 medium (Chu et al.1975) while anther response of Kurulu Thuda wastested in addition on a second callus induction medium,SK-I (Raina and Zapata 1997), as a surplusof anthers were available from this variety duringthe culture phase. N6 and SK-I media were supplementedwith 100mg L -1 yeast extract and containedAgNO 3 (10mgL -1 ) as an ethylene inhibiting agent todelay anther senescence (Lentini et al. 1995). Tworeplicate plates were maintained for each representativestage of panicle maturity in each variety. Aminimum of 318 and a maximum of 616 antherswere cultured per maturity stage depending on antheravailability. Cultures were incubated in thedark at 25 0 C for callus induction.Callus produced on anthers were allowed todevelop to a size of about 3mm in diameter beforetransferring to regeneration medium. Shoot regenerationwas tested on SKH II medium (Raina andZapata 1997) contained in Petri dishes, with eitheragar or agarose as the solidifying agent. When agarosewas used as the gelling agent, the initial transferof callus was onto a hard medium containing1% agarose for subjecting the callus to water stress.After two weeks, the stressed callus was moved onto a softer medium with 0.4% agarose and maintaineduntil regeneration occurred. The growthregulators NAA, BAP, and kinetin were used, eachat 1mgL -1 in the regeneration medium. Two othergrowth regulator concentration regimes were alsotested where either NAA or kinetin concentrationwas reduced to 0.5mgL -1 while BAP concentrationremained at 1mgL -1 . Shoots that were regeneratedon SKH II medium in Petri dishes were transferredonto fresh agar-solidified media in test tubes to allowshoot elongation. Shoots which produced rootswere removed from the culture tube and the rootsystem was washed well in water. The plants with aweak root system were initially nurtured onHoagland liquid medium and kept in the laboratoryfor 2 weeks, before moving into pots with soil andplaced in the glass house.RESULTSPollen stagingAnthers of young panicles of Kurulu Thuda, harvestedwhen the distance between the flag leaf andpenultimate leaf auricle was 3 and 4cm, containedonly uni-nucleate pollen irrespective of the spikeletposition within the panicle from which the antherswere obtained. When the above distance was 5 and6cm, anthers within the panicles had both uni- andbi-nucleate pollen with a higher percentage of binucleatecells in anthers of florets from the upperpart of the panicle. Only bi-nucleate pollen wasobserved in anthers from any part of the paniclewhen harvested from boots that correlated to 7 and8cm of the morphological marker distance.Callus induction from cultured anthersCallus development was observed 4 and 8 weeksafter anther plating in Kurulu Thuda and Bg 250,respectively. In Kurulu Thuda, mainly anthers frompanicles corresponding to the marker distance of 7and 8cm produced callus (Fig. 1a). In Bg250 callusinduction was observed only from anthers collectedfrom panicles when this distance was 6 and 7cm(Fig. 1b). This indicated that the best responsivepollen for Kurulu Thuda were those in the binucleatestage. If the same correlation held forBg250 then the most appropriate pollen would beof the late uni-nucleate or early bi-nucleate stages,but this has not been verified in this study. In KuruluThuda, frequency of callus induction washigher in anthers harvested from panicles at the8cm distance (17.2%) compared to a much loweranther response (3.6%) relating to 7cm distance. InBg250, callus induction frequency was only 1.4%in anthers from panicles of 6cm distance and evenless (0.7%) for those from 7cm range (Table 1).Callus induction was better on N6 medium (3.6–17.2%) than on SK-I medium (0.6–3.3%) in KuruluThuda (Table 1). Callus produced in both varietiesappeared creamy white in colour and friable in texture.Plant regeneration from induced callusFurther proliferation of callus occurred on the regenerationmedium (Fig. 1c). Shoot regenerationwas indicated by the appearance of yellow-greencenters in the callus. Regeneration occurred in callusof Kurulu Thuda only, and this too from callus

Tropical Agricultural Research & Extension 12(2): 2009Table 1: Anthers producing callus from panicles harvestedat different stages of maturity on N6 and SK-Imedia, and the number of regenerated green plantsRicevarietyKuruluThudaCallusinductionmedium*Paniclematurity(cm)N6 6.07.08.0SK-I 6.07.08.0Bg 250 N6 6.07.08.0No. ofantherscultured365616542322546379430594318002293021809060400No. ofCallusinganthersFrequencyofcallusinductionNumber ofgreen plantsregenerated0.03.617.2 12.00.63.32.4 -1.40.70.0 -*Panicle maturity estimated by the distance between the auricles offlag leaf and penultimate leaf at panicle harvestinduced on N6 medium. A total of 115 calli weretransferred from N6 callus induction medium toSKH II regeneration medium. However, the overallanther culture efficiency was poor and only 2 calliproduced green shoots (Fig. 1d), while albinoshoots were regenerated from 5 calli (Fig. 1e). Itwas not possible to assess the effect of growthregulator concentrations and media solidifyingagent on regeneration potential due to the smallnumber of calli showing morphogenetic competence.Clumps of green shoots developed rootswhen moved onto media in test tubes (Fig. 1f).Twelve fully regenerated green plants of KuruluThuda were transferred to pots in the glass house.frequency of indica variety Kurulu Thuda was distinctlybetter on N6 than on SK-I medium. Thissuggests that while some generalization on mediarequirements for the two ecotypes is possible, requirementsof a particular variety within a subspeciesmay be specific for that genotype. Callus inductionfrequency observed for Kurulu Thuda canbe considered reasonably high by indica rice standards.However, manipulation of media componentscould yield further improvements.Callus induction and plant regeneration areconsidered as two distinct phases in the anther cultureprocess of rice. Also it has been reported thatthe rice cultivars that display high callusing abilityshow the best regeneration frequencies (Javed et al.2007; Shahnewaz et al. 2003; Yan et al. 1996). Theresults obtained were consistent with this trend;callus induction was more successful in KuruluThuda and shoot regeneration also occurred fromthe same variety only. However, there are occasionsin which genotypes that show high callus inductionhave displayed poor regeneration abilityand vise versa (He et al. 1998; Talebi et al. 2007).Shoot regeneration in Kurulu Thuda occurred fromcallus that was induced on N6 medium after transferto SK-II medium, but no regeneration occurredfrom callus transferred from SK-I induction medium.This indicates that the callus induction mediumhas an influence on the morphogenetic com-53DISCUSSIONThe distance from flag leaf to penultimate leaf auricle,a convenient morphological marker that hasbeen previously used for estimating the maturitystage of pollen in different rice varieties (Bishnoi etal. 2000) could also be used as a visual guide in theindica variety, Kurulu Thuda.A clear difference in the anther culture responsewas observed in the two varieties of rice.The highest anther response (17.2%) in KuruluThuda was produced from the most mature anthersexamined (from panicles that correlated with the8cm distance). This was over 12 times better thanthe best response (1.4%) observed for Bg250. Similargenotype specificity of anther response withinthe indica subspecies has been observed to be common(Lentini et al. 1995; He et al. 2006; Shahnewazet al. 2003; Talebi et al. 2007).N6 proved to be the better medium for callusinduction in Kurulu Thuda. However, N6 mediumwhich contains inorganic nitrogen in the form ofnitrate and ammonium ions is generally preferredfor japonica rice anther culture. Media in whichnitrogen in the ammonium form is considerablylow or absent, such as the SK-I, are considered betterfor indica varieties (Raina and Zapata 1997).However, in this experiment the callus inductiona b cd e fFigure 1: Callus induction and plant regenerationfrom cultured anthers of indica rice varieties. a-anthercallus of Krulu Thuda; b-anther callus of Bg 250; c-proliferating callus of Kurulu Thuda; d-regeneratedgreen shoots; e-regenerated albino shoot; f–rooted plantlets

54petence of the induced callus, determining its regenerationcapability. Therefore, media manipulationsshould target the production of embryogeniccallus with good regeneration ability rather thansimply inducing prolific callusing, from which regenerationwould not be possible (Silva 2010).Of the seven sub-cultured calli from whichplant regeneration occurred, five gave rise to albinoplants and only two produced green shoots. Albinoplant regeneration is a problem that is commonlyencountered in rice anther culture that diminishesthe utility of the technique as a supplementarybreeding tool for rice.CONCLUSIONSGenotypic differences clearly influence the antherculture ability of rice, and local rice variety KuruluThuda has good anther culture potential. Pollenstaging could contribute to the enhancement of antherculture efficiency by correctly identifying thematurity stage of anthers in each variety prior toculture. The N6 medium was found to be adequatefor callus induction in Kurulu Thuda. Further improvementsmay be possible by manipulation ofmedia components. However, anther culture efficiencymeasured in terms of the green plant regenerationability was poor. Regeneration has to beaddressed with detailed attention to media and cultureconditions that would improve green plant productionand lessen albino plant regeneration.REFERENCESTD SILVA AND WJ RATNAYAKE : ANTHER CULTURE OF INDICA RICEBishnoi U, Jain RK, Rohilla JS, Chowdhury VK,Gupta KR and Chowdhury JB 2000 Antherculture of recalcitrant indica x Basmati ricehybrids. Euphytica. 114: 93 – 101.Brar DS and Kush GS 2006 Cytogenetic manipulationand germplasm enhancement of rice(Oryza sativa L.). In: Genetic Resources, ChromosomeEngineering and Crop Improvement –volume 2, cereals. Ed. Singh RJ, Jauhar PP.CRC press. pp115-158.Chu C, Wang C, Sun C, Hsu C, Yin K and Bi F1975 Establishment of an efficient medium foranther culture of rice through comparative experimentson the nitrogen sources. Sci. Sin. 18:659 – 668.Gupta HS and Borthakur DN 1987 Improved rateof callus induction from rice anther culture followingmicroscopic staging of microspores iniron alum-haematoxylin. Theor. Appl. Genet.74: 95 – 99.He P, Shen LS, Lu CF, Chen Y and Zhu LH 1998Analysis of quantitative trail loci which contributeto anther culturability in rice (Oryzasativa L.). Mol. Breed. 4: 165 – 172.He T, Yang Y, Tu SB, Yu MQ and Li XF 2006 Selectionof interspecific hybrids for anther cultureof indica rice. Plant Cell Tiss Organ Cult86: 271 – 277.Jahne A and Lorz H 1995 Cereal microspore culture.Plant Science.109: 1 – 12.Javed MA, Ishii T, Kamijima O and Misoo S 2007The role of alternating culture temperatures andmaltose in enhancing the anther culture efficiencyof salt tolerant inidca rice (Oryza sativaL.) cultivars, Pokkali and Nona Bokra. PlantBiotechnology. 24: 283 – 287.Kumari HMPS, Silva UND, Abayarathne WM,Abeysiriwardena DS De Z, YatawaraIWMKNK and Sirisena DN 2006 Annals of SriLanka Department of Agriculture:137 – 145.Lentini Z, Reyes P, Martinez CP and Roca WM1995 Androgenesis of highly recalcitrant ricegenotypes with maltose and silver nitrate. PlantScience. 110: 127 – 138.Raina SK and Zapata FJ 1997 Enhanced anther cultureefficiency of indica rice (Oryza sativa L.)through modification of the culture media.Plant Breeding.116: 305 – 315.Raina SK, Balachandran SM, Virmani SS and ZapataFJ 1989 Improved medium for efficientanther culture of some indica rice hybrids. InternationalRice Research Newsletter. 14: 1.Ratheika V and Silva TD 2007 A study on the antherculture response in different varieties ofrice (Oryza sativa L.) sub species indica. Proceedingsof the 27 th Annual Sessions of the Instituteof Biology, Sri Lanka.Shahnewaz S, Bari MA, Siddique NA, Khatun N,Rahman MH and Haque ME 2003 Induction ofhaploid rice plants through in vitro anther culture.Pakistan Journal of Biological Sciences. 6(14):1250 – 1252.Silva TD 2010 Indica rice anther culture: can theimpasse be surpassed? Plant Cell Tissue andOrgan Culture. 100(1):1 – 11.Silva TD and Achala HHK 2008 Microspore stagingfor haploid cell culture in Oryza sativa subspeciesindica (rice). Sri Lnaka Association forthe Advancement in Science. Proceedings ofthe 64 th Annual SessionsTalebi R, Rahemi MR, Arefi H, Nourozi M andBagheri N 2007 In vitro plant regenerationthrough anther culture of some Iranian localrice (Oryza sativa L.) cultivars. Pakistan Journalof Biological Sciences. 10(12): 2056 –2060.Yan J, Xue Q and Zhu J 1996 Genetic studies ofanther culture ability in rice (Oryza sativa).Plant Cell Tiss Organ Cult. 45: 253 – 258(1996).