Relationship between Callus Size and Plant Regeneration in Rice (Oryza saliva L.) Anther Culture

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1 JARQ, 1-1 (1) Relationship between Calls Size and Plant Regeneration in Rice (Oryza saliva L.) Anther Cltre Taiji YOSlDA * Department of Crop Breeding, National Agricltre Research Center (Tskba, baraki, Japan) Abstract Relationship between calls size and plant regeneration in rice anther cltre is described. There was no relation between the calls size and green plant regeneration. Regeneration of albino plants increased in proportion to the calls size in Nipponbare. The nmber of haploid green plants regenerated from callses increased when the calls was>. mm in diameter. Spontaneos dobling of the chromosome nmber of green plants was promoted when the calls was mm in diameter. The ratio of green plant regeneration (green plant/albino plant) and spontaneos dobling freqency of the chromosome nmber of green plants were higher in Akenohoshi than in Nippon bare, which may be related to the fact that the calls sizes of Akenohoshi were relatively small compared with those ofnipponbare. The effect oflow temperatre treatment of anthers on calls formation, regeneration of green and albino plants from callses, and spontaneos dobling of the chromosome nmber of regenerated pl ants was also described. temperatre treatment of anthers reslted in the increase of the calls formation freqency in Nipponbare, bt not in Akenohoshi, as well as the increase of green plant regeneration and decrease of spontaneos dobling of the chromosome nmber of green plants, while low temperatre treatment did not affect albino plant regeneration. Discipline: Biotechnology Additional key words: albino plant, green plant, ploidy level, pretreatment ntrodction Rice tisse cltre has been applied for rice improvement. Especially, anther cltre has been applied to develop new varieties as a simple and time-saving method. Also, transformation and mtation have been applied to introdce new characters in diploid cells. By these methods the gene can be introdced into only one chromosome. As a reslt, the prodction of homozygos plants can not be achieved directly. Therefore, the development of a new cltivar throgh these methods is time-consming or laborios in the same way as throgh ordinary breeding methods. On the contrary, transformation and mtation of haploid cells followed by chromosome dobling and plant regeneration enable to develop homozygos plants. Callses derived from rice anthers show varios ploidy levels ranging from haploidy to high polyploidy, even if they are not treated by chemicals for dobling the chromosome nmber 1 >, and plants with varios ploidy levels can be obtained >. Spontaneos dobling of the chromosome nmber is preferable to the prodction of new varieties by directly sing anther cltre of Fi, or progenies of F1. However, spontaneos dobling before gene indction or mtagen treatment is not preferable to the transformation or mtation methods of haploid cells of anther. Therefore, if green plants can be obtained efficiently and the dobling of the chromosome nmber can be controlled, anther cltre cold be sed for the rapid development of tre breeding lines of rice. There have been several reports dealing with the relation between the calls size and regeneration of green and albino plants in rice >. The problem is to determine whether the calls size is related to the spontaneos dobling of the chromosome nmber. n the present paper, mainly the relationship Present address: Department of Crop Breeding, Chgok National Agricltral Experiment Station (Nishifkats, Fkyama, Hiroshima, 1 Japan)

2 1 between the calls size and plant regeneration was described in rice anther cltre. The effect of low temperatre treatment of anthers on calls formation and plant regeneration was also otlined. Materials and methods Oryza saliva L. cltivars, Nipponbare Uaponica cltivar) and Akenohoshi (cltivar derived from F1 sexal cross between japonica and indica) were sed. They were grown in a glasshose dring the smmer of 1. Panicles were harvested when the base of the flag leaf was - cm above the base of the next lower leaf. The panicjes were not sterilized. N mcdim >, modified to contain. mg/ NaMo H,. mg/ CS H,. mg/ CoCh H and mg/ myo-inositol, spplemented with 1-s M dicblorophenoxyacetic acid (Exps.,,, ) or a-naphthaleneacetic acid (NAA) (Exp. ) and % scrose, was sed for calls formation. The ph of the media was adjsted to. with KOH and.% (Exps.,,, ) or.% (Exp. ) agar was added, then the media were atoclaved al kg/cm and 11 C for 1 min. Twenty anthers were placed in a dish cm in diameter (1 ml medim). Anthers on the medim were treated at 1 C (Exps.,,,, ) and C (as a comrol, only Exp. ) in the dark for O days after placement. They were then cltred at C in the dark. Hndred and forty anthers were cltred for each treatment per cltivar. Calls formation freqency was expressed as the percentage of anthers that formed callses after days of cltre (inclding dration of low temperatre treatment of anthers). For plant regeneration, N modified medim containing OJo agar withot hormones (1 ml in a cm diameter dish) was sed and incbation was condcted at C nder continos illmination with white florescent light ( µes- 1 m- ). After days of cltre (inclding dration of low temperatre treatment of anthers), - O pieces of callses, more than. mm in diameter, were transferred to the regeneration medim in a cm petri dish. The plam regeneration freqency was expressed as the percentage of regenerated callses after days of cltre. After days of cltre for regeneration, plantlets, more than O cm in height, were transplanted into pots comaining soil. Plantlets, less than 1 cm, were again transferred to the regeneration medim, and they were then transplamed to pots if their plant height exceeded 1 cm. The regenerated plants were grown in a glasshose in the winter of 1-1. JARQ () 1 The ploidy levels of the green plants regenerated from anther-derived callses were identified based on their morphological charaeteristics > from November 1 to April 1 as follows: the plants that were shorter with a smaller glme and sterile, as haploids; the plants which had the same morphology and fertility as those of the original cltivars, as diploids (dobled haploids); the plants with larger glme, often having an awn, fertile or sterile, as polyploids or aneploids. Freqency of haploid, dobled haploid, and polypoid + aneploid plants was estimated as follows: (nmber of callses prodcing haploid, diploid, and polyploid + ancploid plants, respectively) /(nmber of callses regenerating green plants) X JQO. Reslts and discssion temperatre treatment of anthers of Nipponbare was effective for calls formation which was 1. times more active than that of the control. n the anthers of Akenohoshi, no effect of the treatmem was observed (Fig. ). Hndred and one varieties of japonica and fifty-for indica varieties were tested (npblished data). The effect of the treatment was prononced in all the japonica varieties, bt not appreciable or not observed in indica varieties. Therefore, the ind/ca traits may be responsible for the lack of the effect of low temperatre treatment on calls formation in Akenohoshi. temperatre treatment of anthers increased the percentage of regeneration of green plants from callses, while no effect of the treatment on the percentage of albino plant regeneration was observed (Table!). ncrease in the percedlage of green plant i. E ().E -;; "o,:) C/) c.. S l!, 1 Fig.. Nipponbarc Treated nl C Not treated ( (') /\kcnohoshi Effect of low temperatre treatment of anthers on the formation of callses from rice anthers Bars indicate standard errors.

3 1'. Yoshida: Relatio11ship be1wee11 Calls Siw and P/111 Regeneration in Rice /1111her Cltre 1 Table l. Relationship between calls size and regeneralion of green and albino plants Cltivars Treatment of anthers Exp. Calls diameter (mm) < < No. of callses tested 1 11 No. of callses Green Albino Plam a) Percentage of callses Green Albino Plani > Nippon bare temp. Exp. ( JOOC) < 1. l < Exp. < l < Exp. + Exp. + Exp. < 1. l < ] J _ < Exp ( C) < < < temp. Exp oo c> Ake nohos hi ( C) Exp. < < a): Plant = Green plants + Albino plants + Dead plants. 1 ll regeneration in the low temperatre treatment of anthers is in agreement with the report of Zho and Cheng >. Akenohoshi exhibited high ratios of green p lant regeneration (green plant/albino plant ratios were.1 in the low temperatre treatment and. in the absence. of treatment, while tj1ose of Nipponbare were. and., respectively). Table also shows the relationship between the calls size and percentage of plant regeneration. The percentage of green plant regeneration from callses, less than 1. mm in diameter, was low nder low temperatre treatment of anthers of Nipponbare (Exp. ). However, the reslts were not. reprodcible in repeated experiments (Exps., ). As the calls size increased p LO. mm in diameter, the percentage of regeneration of albino plants increased. n Akenohoshi, the relationship bet.ween the calls size and plant regeneration was not clearly revealed

4 1 JARQ () 1 becase most of the callses were less than 1. mm in diameter. Wakasa 1 and Ozaki > reported that smaller callses displayed higher abilities of green and albino plant regeneration. These observations were different from the data reported in the present paper. The size of the callses sed by the former athors was relatively large (diameters reported by Wakasa > ranged from - mm and - l O mm; those reported by Ozaki > were in the range of < mm, - mm and > mm). The callses they sed wold be too large to observe a relationship between the calls size and plant regeneration ability since large callses arc old, which res.lts in the redction of celllar activity and indces a decrease of the regeneration ability of green and albino plants. Haploid regeneration freqencies increased by low temperatre treatment of anthers in Nipponbare and Akenohoshi (Table ). When anthers were not Table. Relationship between calls size and ploidy level of regenerated plants Cltivars Treatment of anthers Calls diameter (mm) No. or callses tested No. or callses > HP DHP PAPb> Percentage or callses HP DHP PAPbl < < l Nippon bare temp. E!(P (1 C) < < 1 O J Exp. < < 1 ] Exp. + Exp. + Exp. < < ( C) temp. (JOOC) Exp. Exp. < < < < J Akenohoshi < 1. l Exp ( C). < 1... a): Plants with two or three ploidy levels were regene.rated in some callses. b): HP; Haploid, DHP; Dobled haploid, PAP; Polyploid or Ancploid. c): Reslts of Exp. in low temperat.re treatment of Nippon bare inclded the reslts of Exp. and Exp..

5 T. Yoshid": Relalionship be/ween C"lls Si,e and Plan/ Regenera1ion in Rice An/her C11//11re 1 sbjected to the low temperatre treatment, dobled haploid freqency increased in Nipponbare and the freqency of regeneration of polyploid + aneploid plants increased in Akenohoshi (Table ). n these reslts, low temperatre treatment of anthers is considered to sppress the spontaneos dobling of the chromosome nmber, which is in agreement with the observation of Tsay and Chen >. Spontaneos dobling of the chromosome nmber occrred more freqently in Akenohoshi than in Nipponbare. Table also shows the effect of the calls size on the ploidy levels of regenerated plants. n Nipponbare, haploid regeneration freqency was low in smaller callses (1.-1. mm in diameter) bt increased in larger callses (>. mm in diameter) (Exp. ). On the contrary, the dobled haploid regeneration freqency was high in smaller callses (1.-1. mm in diameter) and decreased in larger callses (>. mm in diameter) (Exp. ). The same relationship was observed when 1.% agar was sed instead of.% agar (Exp. ) or when NAA was sed as the hormone (Exp. ). n total, in the low temperatre treatment of anthers of Nipponbare, haploid regeneration freqencies were.% when callses 1.- l. mm in diameter were sed and.o/o when callses >. mm in diameter were sed; dobled haploid regeneration freqencies were. and.%, respectively. n Akenohoshi, spontaneos dobling of the chromosome nmber mostly occrred in regenerated plants: regenerated plants were mostly derived from callses mm in diameter. These observations may be ascribed to the fact that a calls with a slow growth shows a high potential of spontaneos dobling of the chromosome nmber, or that a calls with late generation displays a high potential. Frther stdies arc reqired to clarify these aspects. n conclsion, when smaller callses (1.-1. mm i n diameter) were transferred to a regeneration medim, dobled haploid plants cold be obtained efficiently. lf larger callses (>. mm in diameter) are transferred to a medim, haploid plants can be easily obtained. References ) Chen, C. C. & Chen, C. M. (1): Changes in chro mosomc nmber in microspore calls of rice dring sccessive sbcltre. Can. J. Gene,. Cytol.,, -1. ) Ch, C. C. et al. (1): Es tablishment of an efficient medim for anther cltre of rice throgh comparative experiments on the nitrogen sorces. Sci. Sin., 1, -. ) Nishi, T. & Mitsoka, S. (1): Occrrence of varios ploidy plants from anther and ovary cltre of rice plant. Jpn. J. Ge11e1.,, 1 -. ) 11, K. (1): Prodction of haploid plants or rice (O,yw sativa) by anther cltre and their se in breeding. 111/. Natl. nst. Agric. Sci.,, 1- [n Japanese with English smmary]. ) Ozaki, K. ( 1); Effects of each factor to calls forming rate and regeneration rate in rice anther cll.ure.. Effects of each factor to regeneration rate. Kinki Chgok Agric. Res., 1, - 1 [n Japanese). ) Tsay, H. S. & Chen, L. J. (1): The effects of cold shock and liqid medim on calls formation in rice anther cltre. J. Agric. Res. Chi11a, (1), -. ) Wakasa (1); Application of tisse cltre in plant breeding. Method improvement and mtant prodction. Bll. Natl. ns/. Agric. Sci., D, 11- [n Japanese with English smmary). ) Zho, X. T. & Cheng, Q. L. (11): The coldpretreatment effect on the rate of pollen-indced plant of hsien rice. J. Fjia11 Agric. College,, -. (Received for pblication, October, 1)