Haploid Plant Production by Anther Culture in Carrot (Daucus carota L.)

Transcription

1 J. Japan. Soc. Hort. Sci. 62(3) : Haploid Plant Producti by Anr Culture Carrot (Daucus carota L.) Kai L Hu, Sachiko Matsubara Kenji Murakami Faculty Agricultural Sciences, Okayama University, Tsushima, Okayama 700 Summary Immature anrs carrot (Daucus carota L.) cultured Murashige Skoog solid ctag various combatis phytohormes. Calli obtaed ctag 0.0~ mg Eliter - 0~ mg Eliter- ket. highest rate callus mati (2%) obtaed ctag mg Eliter- both ket. Embryoids obtaed ctag mg Eliter - 0~0. mg Eliter- ket. highest rate embryoid mati (5%) obtaed ctag mg Eliter- without ket. Embryoids transferred to solid MS grew plantlets. Plants planted to pots after acclimatizati chromosome number root tips observed. Amg 8 obtaed, 6 haploid (2n=9), or two aneuploids (2n=0 ). Introducti Producti new cultivars by hybridizati is tedious carrot, because carrot flowers are small to be ctrolled. Several advantages plant breedg are given with haploid, cludg rapid producti homozygous les as well as possibility to detect recessive mutatis. Sce first paper pollen embryogenesis anr culture Datura noxia by Guha Maheshwari (964), anr culture has been attempted to produce haploid many plant species (Bajaj, 983). Although anr pollen cultures have been permed successfully breedg several plant species, mati haploid carrot by anr or pollen culture has not been reported. This study reports effects phytohormes (ket ) embryoid callus mati carrot anr culture producti haploid carrot. Materials Methods. Embryoid callus mati Anrs carrot 'Senkou 5 sun' grown field Okayama University durg May, 99 Received publicati September 992. used culture. Immature anrs about mm diameter ctag pollen gras at unucleate stage collected morng. y surface-sterilized 0 sec 70% ethanol, immersed sodium hypochlorite ( % available CI) 0 m, rsed three times with sterile distilled water. basal csisted Murashige Skoog (962) with 30 g Eliter - sucrose 2 g Eliter - Gelrite. A factorial design with 6 combatis phytohormes (0, 0.0, 0. or mg Eliter - ket, Table ) employed. ph adjusted to 5.8, autoclaved 5 m at 20 Ž. About 20 anrs plated 5 ml a petri dish (6cm diameter), 5 dishes sealed with Parafilm used each treatment, ree a- bout. 00 anrs used each treatment. Cultures cubated at 25 Ž under 6 hr day length provided with 20ƒÊmol Esec - Em- 2 fluorescent light 35 days. 2. Acclimatizati plantlets Embryoids med carrot anrs transferred to basal cultured 30 days. After roots med, plantlets transplanted plastic boxes with vermiculite watered with /2-strength MS salts every or day 30 days. y n 56

2 562 K. L. Hu. S. Matsubara transplanted to pots, placed field K. Murakami anrs that 60 days. 3. Observati cells chromosome Eighteen tips stored 24hr, 3 Ž water staed 5 3 different 24hr, with : 00 to fixed ket (Table highest (2%) mg Eliter- both ). plantlets 0 50% m anrs. 9 Acclimatizati 3 ctag tip origated anrs, between which different dissected 5 origated used root 2. numbers produced Root plastic : 00, pots roots boxes, transferred multiple all to shoots 3). plantlets After basal some acclimatizg grew normally 4). FAA acetocarme. Results I. Embryoid Embryoids 0, 0.0 or after 0. obtaed ). used quency embryoid as anrs that (5%) without ctag y each 0.0 mg Eliter- ket, Table. Effects anrs. fre- mg obtaed 00 week percentage ctag Calli a mati ket. days About n callus 35 with 2). treatment, percentage. produced after enlarged anrs ket mg Eliter- mati counted mg Eliter- embryoid mg Eliter- ctag anrs mati (Table callus percentage phytohorme highest liter 0~ ccentratis. Embryoids derived a carrot ter 35 days culture. callus embryoid mati carrot anr af-

3 563 J. Japan. Soc. Hort. Sci. 62(3) : Growth an embryoid a carrot derived 4. A carrot plant growg a pot. anr after 42 days. 5. Chromosomes (2n = 9) a root tip cell a haploid carrot plant. or two aneuploid (2n = 0 ). Haploid smaller than diploid es, male sterile. Discussi 3. Multiple plantlets e carrot embryoid cultured MS 30 days. 3. Observati chromosome numbers cells root tip For 6 8 or, chromosome number (2n = 9) observed root tip cells 5); This is first report producti haploid by anr culture carrot. results obtaed present study dicated that origated microspores, because most haploids like as those tobacco anr cultures (Horner et al., 978; Aruga Nakajima, 985). All 5 direct haploids. Two-thirds, however, aneuploids. Formati direct microspores, ree, can be recommended obtag carrot haploid. compositi ccentra-

4 564 K. L. Hu, S. Matsubara K. Murakami tis phytohormes are important factors determg not ly embryoid mati anrs but also subsequent regenerati plantlets. Pollen embryogenesis can be duced a simple meral-sucrose some like tobacco (Nitsch, 969) Hyoscyamus (Raghavan, 975), yet rogenesis to be completed or, supplements with certa phytohormes are required. For stance, cereals, eggplant pepper anrs require both auxs cytoks (Clapham, 977; Matsubara et al., 992). High ccentrati is usually effective callus mati, is hibitory plant regenerati callus. Carrot anr, however, required ly rogenesis. Optimal ccentratis different each or callus direct embryoid mati, amg carrot, eggplant pepper (Matsubara et al., 992). Complex enriched with auxs such as encouragg mati callus should be usually avoided, but present study carrot anr culture, callus mati more promoted than embryogenesis at lower ccentratis. High frequency embryogenesis carrot anrs obtaed with ly mg Eliter-, but callus mati duced with 0.0 to mg Eliter-. Embryogenesis eggplant anrs required essentially high temperature treatment combed with ket (Matsubara et al., 992). rmal shocks given to could alter mode divisi microspore nucleus (Bajaj, 983). rmal shock, however, effective or hibitory carrot pepper microspores by prelimary study (unpublished). Divisi microspore nucleus carrot may be duced ly with phytohormes. Factors ducg embryogenesis microspores differ dependg plant species. For regenerati adventitious shoots,, cultures should be transplanted to basal without phytohormes. frequencies plant regenerati survival after acclimatizati high, acclimatizati very easy carrot. additi aux, especially, to appeared to fluence ploidy (Gleddie et al., 986). Embryogenesis carrot microspores promoted with, subsequent growth normal all haploids. On or h, it is very terestg that twothirds via callus aneuploids that may be duced durg callus mati. Morphological characateristics haploid comm many species. Haploids possess ly e set alleles at each locus, so it is possible recessive mutants to be detected. Furthremore, breedg carrot aims to establish havg cold- heat-resistance, high quality such as high carotee ctent, short root easy mechanizati post-harvest miroot fresh eatg. Doublg chromosome number haploids fers a method rapid producti homozygous. Literature Cited Aruga, K. T. Nakajima Role anr pollen embryogenesis anr culture Nicotiana tabacum L. Japan. J. Breed. 35 : Bajaj, Y. P. S In vitro producti haploids. pp In: D. A. Evans, W. R. Sharp, P. V. Ammirato Y. Yamada. (eds.). Hbook plant cell culture. Vol.. McMillan, New York. Clapham, D Haploid ducti cereals. pp In: Reert, J. Y. P. S. Bajaj (eds.). Applied fundamental aspects plant cell, tissue, organ culture. Sprger-Verlag, Berl, Heiderberg, New York. Gleddie, S., W. A. Keller G. Setterfield Eggplant. pp In: D. A. Evans, W. R. Sharp P. V. Ammirato (eds.). Hbook plant cell culture. Vol. 4. McMillan, New York. Guha, S. S. C. Maheshwari In vitro producti embryos anrs Datura. Nature 204 : 497. Horner, M. H. E. Street Pollen dimorphismorig significance pollen plant mati by anr culture. Ann. Bot. 42 : Matsubara, S., K. Hu K. Murakami Embryoid callus mati pollen gras eggplant pepper by anr culture. J. Japan. Soc. Hort. Sci. 6 : Murashige, T. F. Skoog A revised rapid growth bioassays with tobacco tissue cultures. Physiol. Plant. 94 : Nitsch, J. P Experimental rogenesis Nicotiana. Phytomorphology 9 : Raghavan, V Inducti haploid anr culture henbane. Z. Pflanzenphysiol. 76 :

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