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•i IR !RESEARCH PAPER'.ESERIES NUMBER 79 .AUG UST 1982

SEED-DER ED CALLUS' CULTURE FOR SELECTING SALT-TOLERANT

PART I CALLUS INDUCTION PLANT REGENERATION, AND VARIATIONS IN VISIBLE PLANT TRAITS

K. SUENAGA, E. M. ABRIGO, and S. YOSHIDA

The International Research Institute PO. Box 933, Manila, Philippines SEED-DERIVED CALLUS CULTURE FOR SELECTING SALT-TOLERANT hICES. PART I. CALLUS INDUCTION, PLANT REGENERATION, AND VARIATIONS IN VISIBLE PLANT TRAITS 1

ABSTRACT

Seed-derived callus culture was used to study cal- quencies of plant regeneration were higher at lus induction and plant regeneraticn in 15 indica higher levels of sodium chloride in all varieties and virieties. Effects of different tested. concentrations of scdium chloride in the selection medium on callus growth and subsequent plant re- Variations in visible plant traits were observed generation were also examined. Varieties differed in the regenerated plants (RI) and te subsequent markedly in their ability to induce callus and to generation (R2). In RI plants of Taichung 65, high regenerate plants. Callus induction frequencies sterility (14.6/100 plants), tetraploids (3.4/100 ranged from 32 to 85% in the absence of yeast ex- plants), and other abnormal traits, such as tract ani casein extract. It was, however, in- stripes, open spikelets, etc. (0.9/100 plants) creased to nearly i)0%when they were added. Plant were observed. In R2 seedlings, the pooled chloro­ regeneration frequencies ranged from 0% for IR8, phyll mutation frequency of 1,259 lines was IR26, IR28, 1R30, IR36, and Koshihikari to 62% for 8.8/100 lines. This value is as high as that with Taichung 65. This frequency decreased as the cal- irradiation but lower than that induced by chemi­ lus culture period was lengthened. Iowever, 27- cal mutagens. week-old callus of Taichung 65 and Nona Bokra re­ tained 30-40% rpreneration ability. Even 47-week- Variations in dwarfism, high sterility, heading old callus of far,-hung 65 were able to regenerate date, brown leaf spots, chlorophyll mutations, and plants. Tie ad('.tion of sodium chloride to the various morphological traiLs were observed on selection medlu., had pronounced effects on callus field-grown R2 plants. About 54% of Taichung 65 R2 growth and subsequent plant regeneration. No cal- lines exhibited variations in 1 or more visible lus growth was observed at 3% sodium chloride, a plant traits; 17.5% in more than I trait. Among few calluses grew slightly at 2%. Callus growth the traits observed for variation, the occurrence was good at concentrations less than 1%. At 1.5% of sterility was most pronounced; about 30% of sodium chloride, callus growth was retarded but total R2 lines of Taichung 65 had less than 75% moderatc growth was maintained during the first fertility. The occurrence of sterility in RI and selective passage. With ll-week-old callus, fre- R2 plants did not correlate.

I/By Kazuhiro Suenaga, junior plant breeder, Agricultural Research Center, Kannondai, Yatabe, Tsukuba-gun, Ibaraki-ken 305, ; Editha M. Abrigo and Shouichi Yoshida, research assistant and plant physiologist, IRRI, Los BaRos, Laguna, Philippines. Submitted to the IRRI Research Paper Series Committee May 1982. PREVIOUS

SEED-DERIVED CALLUS CULTURE FOR SELECTING SALT-TOLERANT RICES. PART 1. CALLUS INDUCTION, PLANT REGENERATION, AND VARIATIONS IN VISIBLE PLANT TRAITS

ligh soil salinity is one of the most important regenerated plants. In rice, Oono and Sakaguchi soil constraints to rice productioq in developing (1978, 1980) showed that some plants regenerated countries. in Suth and Southeast Asia, about 54 from callus grown on a salt-enriched medium were million ,i of land are considered sallne soils, tolerant of high salinity and that salt tolerance 50% of them in coastal regions (Akbar and Ponnam- was probably heritable. It is not clear in that peruma 1982). Rice varieties with increased salt work, however, if plant regeneration was made from tolerance would increase and stabilize rice yields salt-tolerant varian t callus lines. Kucherenko in salt-affected areaL such as coastal regions and (1980) stated that there was no relationship be­ canal-irrigated areas. Increased salt tolerance tween the salt tolerance of the whole plant and would also permit ex)ansion of rice cultivation thdt of the callus and that differences between into marginal saline areas. tolerant and susceptible varieties were not visi­ ble at the callus level. Screening existing materials for salt tolerance has had some success (Ikehashi and Ponnamperuma In rice, plant regeneraticn from callus can be 1978). However, salt tolerance needs to be in- made through either anther culture or somatic tis­ creased beyond the maximum level now available, sue culture (Niizeki and Oono 1968, Maeda 1968, Salt tolerance levels appear to be mucl- lower in Nishi et al 1968). However, in anther culture of rice than in other grain crops such as barley and rice, the capacity of callus to regenerate plants wheat (Maas and Hoffman 1977). diminishes so rapidly with passa;-e in subculture that it is totally lost by the second passage Anther culture is evolving as an innovative plant (Oono 1975). This rapid loss of morphogenetic breeding technique that can shorten the time capacity severely restricts use of anther culture required for obtaining homozygosity. Several for induction and selection of mutant plants. This varieties of rice and tobacco have been developed limitation is particularly valid if mutation ana through anther culture (Zen-hua 1980, Nakamura et selection are achieved by repeated cycles of sub­ al 1975). culture under a given stress. Furthermore, anther culture is tedious and produces a large number of Induction and selection of useful mutant plants is albino plants. another promising area where tissue and cell cul­ ture techniques can be util ized effectively. Evi- In somatic tissue culture, seeds are an excellent dence has accumulated to indicate that the tissue material because the vigorous surface steriliza­ culture cycle itself generates genetic variability tion results In minimal contamination of the re­ (Larkin and Scowcroft 1981). sultant callus culture. For those reasons, seed­ derived callus culture was used for this study. Selections have been Made at the cellular level Work began in December 1980 with these for resistance to antibiotics, amino acid ana- objectives: logues, and salts (Cono 1979). Some of that re- 1. Develop an efficient seed-derived culture sistance was manifested in the regenerated plants system, from callus induction to screening and analyzed genetically (Carlson 1973, Marten and of regenerated plants for salt tolerance. aliga 1975, MaLiga et al 1975, Cegenbach et al 2. Increase salt tolerance of improved 1977, Chaleff and Parsons 1978, Bourgin 1978). varieties and traditional salt-tolerant varieties. Several scientists have attempted to increase salt 3. Improve agronomic traits of traditional tolerance of some plant species at both the eel- salt-tolerant varieties. lular and the whole plant level by tissue culture techniques (Croughan et al 197h; l)ix and Street 1975; Dix 1980; Kochba et al 1980; Kucherenko This report focuses on varietal differences in 1980; Nabors L al 1975, 1980; Oono and Sakaguchi callus induction and plant regeneration and 1978, 1980; Stavarek eL al 1980, Zenk 1974). describes the variability in regenerated plants (RI) and their progeny (R2). At present, more knowledge is available on salt tolerance at the cellular than at the whole plant level. Select ion of salt-tolerant plants. Is MATERIALS AND METIIODS limited to only two species: Nicotiana tabacum and . Nabors Kt al produced salt-tolerant Varieties variant celi lines of Nicotiana tabacum by ex­ posing cel Is to a saIt-enriched iiquid medium. Seeds of the 15 tndica and japonica varieties used They were able to regenerate salt-tolerant plants. in this study (Table 1) were provided by the Plant They also demonstrated that salt tolerance was Breeding Department of the International Rice heritable, at least to the third generation of Research Institute. 4 IRI'S No. 79. August 1982

Table I. Varieties used in this study. in diameter) and three pieces were placed in each Variety Accession no. (or Origin flask containing S medium. Flasks were kept in st sA our) Odarkness at 28'C. Callus was transferred to iden­

tical S medium at 4-week intervals up to the IR8 10320 Philippines seventh passage. At the end of each subculture IR26 Ryt 1834 Philippines from the third to the seventh passages, a portion IR28 Ryt 1701 Philippines of proliferated callus was transferred to R medium IR30 OIB408 Philippines with one piece of callus/tube for plant regenera- IR36 30416 Philippines tion. Test tubes were kept in light at 28°C. iR42 Ryt 1970 Philippines AI)T28 26829 India Kalarata 1-24 26913 india Culture of regenerated plants (RI) Nona Bokra 22710 India SR26B 590E India Plantlets that reached the mouth of the test tubes Fujisaka 5 244 Japan were transferred to soil. Before transfer, *the Koshihikari 36820 Japan agar was removed from the roots. Then roots were Norin 20 418 Japan soaked in 0.1% nicotinamide aqueous solution for I Reiho 40025 Japan hour to stimulate root development. Plantlets Taichung 65 29 Taiwan originating from 1 callus were planted together in a 1.8-liter pot and placed in a lighted cabinet where high humidity was maintained to keep plants from withering.

Culture media A week later, plantlets were transferred to the glasshouse. About 3 weeks later, they were sep- The culture medium (C medium) for callus induction arated into single plants and individually trans­ and callus proliferation was Murtshtge and Skoog's planted at 1 plant/I.8-liter pot or 3 plants/3.8­ basic salts (Murashige and Skoog 1962) supple- liter pot. About 30 days after f).owering, R2 (the mented with 2 mg 2,4-D/liter of medium. For the next generation of regenerates) seeds were har­ selection media (S media), sodium chloride (0, vested individually and kept in an oven at 50C 0.5, 1.0, 1.5, 2.0, 3.0% wt/vol) was added. The for 5 days to break dormancy. regeneration medium (R medium), with the same basic composition as the C medium was supplemented with 3 g yeast extract, 3 g casein hydrolysate, 5 Investigation of R1 and R2 plants - 5 x 10 mol kinetin, and 2 x 10-6 mol NAA (instead of 2,4-D)/liter. Sucrose was increased to 70 g/ Plants that originated from one callus were desig­ liter. nated as a callus line in both Ri and R2. The pro­ getty of one RI plant was designated as a plant The pil values were adjusted to 5.8 and 8 g agar/ line In R2. liter was added. After the agar was melted, media were dispensed Into 50-ml Erlenmeyor flasks and 25 Differences in planting method, growing time, and x 200 mm test tubes at 20 ml/flask or test tube. weather conditions made it impossible to collect Flasks and test tubes were covered with 2 layers comparable data on agronomic traits among RI of aluminum toil and autoclaved at 120C for 15 regenerates. Observations were made on spikelet mini tes. sterility, tetraploidy, extreme dwarfism, stripes, and abnormal glumes.

Callus induction and callus growth The varieties used for investigations of R2 gener- Seeds were dehulled manually, soaked in 70% ethyl ation were Kalarata 1-24, Nona Bokra, Fujisaka 5, alcohol for I minute, immersed in 0.1% mercuric Norin 20, Reiho, and Taichung 65. All lines were chloride aqueous solution for 20 minutes, and obtained from the third passage with 0, 1.0, and rinsed several times with sterilized distilled 1.5% NaCl. One large panicle from the main culm water. Three seeds/flask of C medium were kept in or primary tillers with high fertility was chosen darkness at 28°C for 3 weeks for callus induction, from each RI plant to ensure about 40 seeds. Seeds Callus induction frequencies were then recorded. were sterilized with saturated calcium hypochlo- Induced calluses we'e separated carefully from the rite solution for 15-20 minutes and incubated in rest and transferred to fresh C medium (3 darkness at 28'C for 2 days. They were sown on pieces/flask) and allowed to prolif. ate for 4 seed boxes 3-5 October 1981 and kept in the weeks under the same conditions. Relative callus glasshouse. growth was recorded at the end of the second passage. On 22 October, 20 seedlins/line were trans­ planted, spaced 25 x 50 cm, , in the field at Calluns cul tureon the selection Masapang, Victoria, Laguna, Philippines. Before medium and plant regeneration transplanting, chlorophyll mutants and other ob­ vious variants were recorded. Conventional culti- Callus that proliferated on C medium for 2 pas- vation procedures were followed. Visual observa­ sages was divided into 50-mg pieces (about 6-8 mm tions of traits were made 1 month before and 1 IRPS No. 79. August 1982 5

month after flowering. Spikelet sterility scores Callus growth on selection medium were taken at appropriate times after flowering (IRRI 1980). The higher tile concentration of sodium chloride, the poorer the callus growth. No callus growth was observed at 3% sodium chloride, few calluses grew RESULTS AND DISCUSSION slightly at 2%, but callus growth was good at con­ certrat ion less than At 1.5Z sodium chloride, callus growth was retarded but moderate during the Callus induction and growth first selective passage. Repeated subculturing on S medium 1 d to even poorer growth, but some cal- The earliest callus formation was observed about 5 luses naintained stable growth throughout the days after inoculation. Callus initiated from selectie passages. No test was made to determine basal regions of the mesocotyl or coleoptile. if those calluses were salt tolerant.

Japonica varieties (Reiho, Norin 20, Taichung 65, Plant regeneration Fujisaka 5, and Koshihikari) and some indica vari­ eties (Nona Bokra and Kalarata 1-24) displayed When sodium chloride wa not added to the S higher callus induction frequencies than did all medium, frequency of plant regeneration from 11- IR lines except IR28 (Table 2). IR lines initially week-old callus ranged from 0" for iRS, IR26, exhibited good callus growth, but most callus, ex- IR28, IR30, IR36, and Koshil.1kari to 62% for cept in IR28, soon stopped growing and turned ne- Taichunig 65 (Table 3). On the average, eech re­ crotic. tIR28 had vigorous callus growth during the generation produce(] about eight plants. Callus of first passage, but its callus developed root-like most IR lines turned necrotic a f, weeks; after organs during the second passage. Adding yeast ex- they were transferred to K medium. Almost 10% of tract (2 g/liter) and casein hydrolysate (2 Koshihikari call us developed roots hut no shoot. g/liter) to C medium increased callus induction Taichung 65, Nona Bokra, and ReLiho showed more vigo­ freauencies of all varieties to nearly 100% and roes cal lus growth and higher plant regeneration. improved callus growth rates considerably. More whitish to pale green alhino-like plants were observed in japonica varieties. Callus was hard and whitish in Kalarata 1-24; mod­ erately hard and yellowish in Nona Bokra, Reiho, Frequencies of albino-like plants were lower in and Taichung 65, soft and whitish in Fujisaka 5, this seed-cutture experimcnt than in anther cul- Koshihikari, and Norin 20. ture, which tntay be an advantage of seed-derived callus over anther-derived callus.

Table 2. Varietal differences in callus induction and subsequent callus growth. Seeds % seeds Table 3. Variet'il differenc2s in plant regenera­ planted tat Callus tion frequencies. plantd id g rowth Varie _u nand inducedatdeb/ Test Frequencies (%) a / unc:ontaminated (no.) callus rate Variety tubes Plant regenera- b/ IR8 147 53.7 1.39 (no.) tions a / Albino­ 1R26 152 42.8 0.81 1R28 98 68.4 4.79S/ IR8 12 0.0 - IR30 132 31.8 0.58 IR26 3 0.0 - IR36 42 57.1 1.14 IR28 4 0.0 - IR42 131 52.7 0.91 IR30 7 0.0 - ADT28 140 40.7 0.99 IR36 6 0.0 - Kalarata 1-24 137 64.2 2.31 1R42 6 16.7 0.0 Nona Bokra 159 63.5 2.55 ADT28 38 15.8 0.0 SR26B 96 53.1 0.98 Kalarata 1-24 71 18.3 7.6 Fujisaka 5 168 70.8 2.45 Nona Bokra 86 53.5 0.0 Koshihikari 132 66.7 2.29 SR26B 18 16.7 0.0 Norin 20 159 75.5 2.37 Fujisaka 5 82 17.0 42.8 Reiho 119 84.9 3.16 Koshihikari 61 0.0 - Taichung 65 158 72.8 2.77 Norin 20 b4 12.5 25.0 Reiho 67 31.3 4.7 a/Observed 3 weeks after inoculation. Those that Taichung 65 131 61.8 13.5 were observed to have formed necrotic call uses were recorded the same way as those that did not form / No. of calluses from which plantlets were reiPne!a­ calluses. b/Scored at the end of the second passage ted : no. of calluses observed x 100. li/I nc ludes by the following scale: I = 2.5 mm in diameter, not only alhinos hut also those thot were low in = 2 = "5.0, 3 .:7.5, 4 = <10, 5 = 10. Those that chlorophyll content (xantha). Computed as no. of produced no calluses, or calluses that turned calluses from which only albino plantlets were necrotic at the scoring time were scored 0. £/Root- regenerated : no. of calluses frfm wh'liich plant lets like organs were observed on most calluses, were regenerated x 100. 6 IRPS No.79, August 1982

As the callus culture period was lengthened, plant Tble 4. Plant regeneration frequencies as affected regeneration frequency decreased in Noia Bokra, b) length of culture period. Reiho, a.,d Taichung 65 but remained almost un- Frequencies (Z) at given changed in Kalarato 1-24 and Fujisaka 5 (Table 4). Fre nof s ature Taichung 65 and Nona Bokra retained 30-40% regene- Va1ety 1wkwkf eriod15 wk callu19 wks culture23 wk 27 wk ration ability in 27-week-old callus. In a sepa­ rate experiment, plants were regenerated from 47- Kalarata 1-24 18.3 13.8 29.6 16.7 15.3 week-old callus of Taichung 65. Reported frequen­ cies of plant regeneration vary considerablv. Nishi Nona Bokra 53.5 58.8 64.7 40.0 39.7 at al (1968) obtained I100A plant regeneration in 2­ month-old japonica variety Kyoto Asahi callus, 91% Fujisaka 5 17.0 12.9 11.4 7.8 12.9 in 4-month-old callus, and 64% in 6-month-old callus. Saka and Maeda (19)69) reported 37Z plant regenera- Norin 20 12.5 6.0 17.5 7.5 6.0 tion in 2- to 2-month-old callus. In a similar study by Hlenke et a! (1978), plant regeneration ire- Reiho 31.3 21.7 25.4 14.2 14.6 quency sharply reduced, from 61Z in 8-week-old callus to 7% in I-.eek-old callus. Differences Taichung 65 61.8 50.8 38.8 25.5 32.9 in plant regeneration frequency are largely attri­ buted to variety, length of culture, and concen­ trations of auxins in the R medium. In haploid Table 5. Plant regeneration frequencies of 11-week callus induced through anther culture, no plant and 27-week-old calluses at different levels of regeneration was observed after the second passage NaCia/ in subculture (Ono 1975). Variety --- -- Frequencies (Z) 0 NaCl 1.0% NaCl 1.5% NaCl Frequencies of plant regeneration by level of Nona Bokra 53.4-39.6 47.6-23.0 61.5- 0.0 sodium chloride in S medium and length of culture are shown in Tabfl 5. With l1-week-old callus, uj isaka 5 17.0-12.9 32.1- 7.5 43.7-11.5 plant regenerat.on frequencies were higher at higher levels of sodium chloride in all varieties Norin 20 12.5- 6.0 20.6-11.6 58.8- 3.3 tested. urashige and Nakano (1967) suggested that reduced regeneration ability was due to increased Reiho 31.3-14.6 28.5-14.2 55.5-27.2 somatic age, which in tun increased aneuploidy of callus celIs in tobacco. This nay explain the in- Taichung 65 61.8-32.9 66.1-38.7 76.9-27.2 creased plant regeneration frequencies with in­ creasiag levels of soditam chloride in S medium. A-/NaCl was added to the selection media but not to Sodium chloride retards cal lIs growth, probably by the regeneration medium. decreasing the rate of cell division, which in turn reduces occurrence oft aneupIoidy and retains a high plant regeneration capacity. With 27-week- 0.9%. Such phenotypic variation in regenerated old callus, the effects of sodium chloride on plants has been reported earlier (Nishi et al plant regeneration frequency were not consistent. 1968, Nishi and Mitsuoka 1969, Niizeki and Oono In Nona Bokra, plant regeneration sharply reduced 1971, Oono 1975, Oono et al 1976, Henke et al at 1.57. 1978). Usually, most genic mutations are consi­ dered to be governed by recessive genes. Some of Rl plants the abnormal characters observed in RI plants, such as high sterility and dwarfness, might be due to chroiosomalI aberratilon. Occurrence of tetra­ i ion Of th t rtp Unusual traits observed In Taichung 65 R1 plants to plaotsoia a ner type regnerated after tie third passage included ab- ploid plants is oer ind[cation of that and var iat ian. normal IUlUes (open spikelets, long glumes, small antd raouid glumes), awns, dwarfs, sterility, Plants originating from the same callus line stripes, mid LeLraploids. Observations were re­ ctcod artetapoius,hih serlit, ad thewvried in tile expression of these characters. In tordefora ale 6). eal h sig i stericeire, and r- other words, plants from the same callus line were tra its (Ta b le 6 ) . Tlet ralp lo id s in rioc e are ch a ra e -d i f r n i n W g e o y . terized by somewha t !;tut Led growthi, fewer pani­ cles, thicker and coarser leaves, thicker culms,

poor panicle ex.ertion, larger grain size, awns, CllorophiyilI lutat ions in _R2syeedlings- and high sterill ty (Morinaga and Fukushima 1937). High sterility refers to less than 5OZ fertilIty. Several kinds of chlorophyll mutants were observed )ther traits Inc lude abnormal glumes, dwarfs, and at the seedling stage of R2 plants: I) albino, 2) stripes on leaf blades mud sheaths. 1lbi no-like lethal yellow (xantha - the color ranged from yel­ plants were excluded. low to pale green and seedlings died within 2-3 weeks), 3) pale green, ond 4) stripes (various About 15% of the R1 plants had less than 50% for- kinds of stripes were observed; the mos, common t ill ty, caused part ly by envi ronmentaL facors. were white Iligitudinal stripes that u; -ginated Tetraploid frequency was 3.4% and obvlius varta- from the tip and developed along the margin of tion,;, such as l ri pes and abnormal glumes, were leaf blades). IRPS No. 79, August 1982 7

Table 7 3alows the frequency of chlorophyll mutants line are not always of the same genotype. Normal­ in R2 seedlings. The pooled frequency of 1,259 ly, most chlorophyll mutations are assumed to be lines was 8.8/100 R2 lines. This value was as high controlled by single recessive genes and ratios as that obtained by irradiation, but lower than are expected to be about 0.25. Ratios for Nona that induced by chemical mutagens. According to Bokra and Taichung 65 in this study were about Mikaelsen et al (1971), frequencies of chlorophyll 21%. mutation by gamma rays (or fast neutrons) and by ethyl-methane sulfonate (EMS) treatment were about 10-30/100 M2 panicles. Much higher frequencies Table 6. Observed variants of Taichung 65 in RI of 40-70/100 ',2 panicles were obtained by treat- plants. ing fort i ized r ice ova with N-me thy I-N­ nitrosourea (NMU) (Satoh and Omur~a 1979). Tissue NaCl concentration Total 50% fer- Tetra- Others culture appears to be a s effective as irradiation (U) tilitv ploid but less offctive tHMn chemi cil nutagens such as 1.5 EMS and NMIU.a illdlCiill chlorophy11ll EMdcis n NU.ali rpy mutaItion.uta h Dif-i~Callus 1 ines (no. ) lb 9 3 3 fert nces in chlorophy 11 mutation frequency among levels of sodium chioric[t were erratic. R1 plaits (no.) 165 22 5 4

Table 8 shows averagt aeg regat.ion ratios of chlo- 1.0 rophyll mutants in I2 lines which segregated mu- Callus lines (no.) 35 10 5 1 tants at the seedlin, stagO. The agregation ratio RI p)lants (no.) 201 18 19 1 of chlorophyl I nutai.t:; obtained from each mutated line varied from 3 to 77/. The lower frequency is 0 aitributed to cii merlam. The higher frequency Callus lines (no.) 58 20 1 2 could be attribu ted t,a double mu taiit , because one1)lantLs.R2 i inc segreattel both al hino and striped To talI'a

Callus lines (no.) 109 39 (35.8) 9 (8.3, 6 (5.5) RI plants (no.) 742 108 (14.6) 25 (3.4) 7 (0.9) Plant lines originiaLin , from the !;a callus line also differed ill aegregat ion ratio of chlorophyll / Numbers in parentheses are percent of variants of mutants. This obsrvat ion supo)rts the earlier the total callus lines or plants. conclusion that R1 p larts from the same callus

T)abl e 7. Frequencies of clo [aropyl l mutants in R2 seedlings of 6 varieties.­ 2ulutatJon f re l ''suc Variety NaCl R2 Muteted R2 r0tated(0 Per1,000 concentration ILies (no.) lines (no.) plants (no.) plants (no.) K2 lines R2 [a ntS

Kalarata 1-24 II 41 1 1,124 1 '2.4 U .9 1.0 17 0 313 0 (.10 0 .0 1.5 11 1 319 13 9.1 '40.8

Nona Bokra 0 185 14 4,494 76 7.6 16.9 1.0 145 11 3,987 44 7.6 11.0 1.5 87 21 2,182 106 24.1 48.6

Fujisaka 5 0 27 1 595 10 3.7 16.8 1.0 38 1 874 7 2.6 8.0 1.5 12 0 274 0 0.0 0.0

Norin 20 0 25 4 632 27 16.0 42.7 1.0 23 0 603 0 0.0 0.0 1.5 27 0 579 0 0.0 0.0

Reiho 0 66 2 1,648 9 3.0 5.5 1.0 7 0 199 0 0.0 0.0 1.5 21 0 440 0 0.0 0.0

'raichung 65 0 245 28 6,508 160 11.4 24.6 1.0 138 11 3,882 61 8.0 15.7 1.5 144 16 4,582 112 11.1 24.4

Pooled 1,259 Ill 33.235 626 8.8 18.8 a/ Observation!; were made about 2 weeks after seeding. 8 IRPS No. 79, August 1982

Variability in field-grown R2 plants Table 8. Segregation ratio of chlorophyll mutation in R2 seedlings. Table 9 summarizes frequencies of some visible NaCl con- Mutated Plants (no.) Segre­ traits, singly or in combination, in R2 lines of Variety centra- plants in mutated gation Taichung 65. A plant was recorded es a dwarf when tion (Z) (no.) lines ratio (%) the plant height of a R2 plant was one-half that of the normal plant or shorter or when more than 3 Nona Bok:a 0 76 342 22.2 plants had heights more than 10 rm less than that 1.0 44 298 14.8 of the normal plants in I line (20 plants) and 1.5 106 414 25.6 when there was no sign of disease and insect dam- Total 226 1054 21.4 age. Late headJng variants were recognized in com­ 20.2 bination with chlorophyll mutation and by at least Taichung 65 0 160 792 19.5 a 10-day delay in heading date. Morphological var- 1.0 61 313 458 24.5 iations inc. ded the appearance of unusual glumes 1.5 112 or grains, anicles, leaves, and tiller arrange- Total 333 1563 21.3 ments (Table 10). When spikelet fertility was less than 75%, a plant was considered a variant in sterility. Abcut 54% of the R2 lines of Taichung High sterility. Fertility of R2 plants ranged from 65 exhibited variations in I or more visible plant 0 to almost 100%; the parent variety showed stable traits. Vari tons in more than 1 trait were exhi- and high fertility (>90%). About 30% of the total bited by 17.5, if R2 lines. R2 lines of Taichung 65 had less than 75% fertil­ ity. Such sterile variants can be attributed to Oono (1979) reported that 72% of R2 lines had such genetic variations as chromosome aberration, variations in tetraploidy, plant height, spikelet cytoplasmic or nuclear changes, and interactions fertility, heading date, morphological traits, and with the environment. The mode of segregation dif­ chlorophyll deficiency, singly or in combination. fered from line to line. Some showed continuous The difference between the 72% of Oono's results variation in fertility, and others had sterility and the 54% of ours may be attributed to A"fferen- variants clearly distinguished from the normal. In ces in variety and the environment under which R2 some cases, high sterility was accompanied by mor­ plants were grown. phological variations such as dwarfness and abnor­ mal panicles.

Table 9. Frequencies of variations in R2 plants of Taichung 65. 1.5% NaCl 1.0% NaCI 0 NaCl Total -- b i Variant R2 FrequencyTN R2 Frequency l R2 Frequencyb/ R2 Frequency-. traits a / lines lines lines lines (no.) (no.) (no.) (no.)

Normal 76 52.4 66 47.9 96 40.3 238 45.6 bl 16 6.7 16 3.1 ch 5 3.4 9 6.5 12 5.0 26 5.0 d 21 14.5 8 5.8 7 2.9 36 6.9 m 2 1.4 4 2.9 13 5.5 19 3.6 s 13 9.0 28 20.4 55 23.2 96 18.3 bl, d 3 1.3 3 0.8 bl, m 2 1.4 3 :.3 5 1.0 ch, d 6 4.1. 10 4.2 16 3.1 ch, 1 1 0.7 1 0.2 ch, m 1 0.7 1 0.2 ch, s 6 4.1 1 0.4 7 1.3 d, m 1 0.7 1 0.7 2 0.8 4 0.8 d, s 10 6.9 4 2.9 10 4.2 24 4.6 m, s 3 2.1 8 5.8 1 0.4 12 2.3 bl, ch, m 1 0.7 1 0.2 ch, d, m 1 0.4 1 0.2 ch, d, s 1 0.7 1 0.7 1 0.4 3 0.6 ch, m, s 3 1.3 3 0.6 d, m, s 4 2.9 1 0.4 5 1.0 ch, d, m, S 1 0.7 3 1.3 4 0.8 Total of varn- 69 47.6 72 52.1 142 59.7 283 54.4 ant trait'; a/hi = brown leaf spot, ch = chlorophyll mutation, d - dwarF, I = late heading, m morphological variation, s = sterility. A/No. of lines which segregated variants : no. of lines observed x 100. IRPS No. 79, August 1982 9

In R1 plants of Taichung 65, about 15% showed less Table 10. Kinds of morphological variations than 50% spikelet fertility. In its progeny lines observed in the field. (R2), 31% had less than 50% fertility. On the other hand, in progeny lines from RI plant lines Plant part Variant traits whoie fertility was higher than 50%, 29% had less than 50% fertility. Although detailed statistical Glume or grain Long and big, small and round, analysis was not done, the results suggest no cor- awned relation between RI and R2 in sterility. Panicle Clustered, dense Leai Rolled, short, nacrow, erect Other visual traits. Other visual traits, includ- Tiller Compact tiller arrangement ing chlorophyll mutation, brown leaf spots, dwarfs, and morphological traits were observed (Table 9). Chlorophyll mutation includes white and yellow stripes. Each trait was manifested singly or in combination with other traits. Frequency of R2 plants originating from 3 callus lines of Nona dwarfs was the second highest (18.6%), next to Bokra headed 3 to 4 weeks later than the parental high sterility (29.5%). About 40% of dwarfs were plants. Only a few plants were found to head accompanied by sterility or morphological varia- earlier than the parental plants. tions. Various kinds of morphological variations observed on Taichung 65 are listed in Table 10. The data collected show that seed-derived callus culture can generate variations in agronomic traits such as spikelet sterility, plant height, Of 13 lines which segregated viable chlorophyll heading date, and panicle type. mutants at the seedling stage and which were transplanted in the field, 7 survived. Five re­ covered from chlorophyll deficiency as growth ACKNOWLEDGMENT advanced.

Heading date. Range of headiiig dates, average We would like to thank Prof. T. Kinoshita, hfeading dates, and their standard deviation are Hokkaido University, Japan, for his constructivc shown in Figure I. Average heading dates of R2 comments on this paper. plants were not much different from those of the parental varieties. But there was a general trend for R2 plants to head later than the parent. Some

Kolarato 1-24 P i R2 ,' -

Nona Bokra

P ',-H Fujisaka 5 R2 i, 0 i

Norin 20 0 = av date of heading R 2 iI I standard deviation

p P = parent variety Reiho R2 z R 2 plants R2 S

Toichung 65 R2 I S I - .... I ... .1 ...... L...... I I 50 60 70 80 90 100 1HO 120 Days (no) from seeding to heading Fig. 1. Range of heading dates, average date of heading, and standard deviation of R2 plants. 10 IRPS No. 79, August 1982

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