HORTSCIENCE 25(10):1276-1277. 1990. Polyembryony in Undeveloped Monoembryonic Diploid Seeds Crossed with a Citrus Tetraploid I. Oiyama and S. Kobayashi Akitsu Branch, Fruit Tree Research Station, Akitsu, Hiroshima, 729-24 Japan Additional index words. embryogenesis, seed development, polyploid Fig. 1. Normal (left) and undeveloped (right) Abstract. Some undeveloped seeds from mature Citrus fruit of monoembryonic diploid seeds from mature fruit of 2x ‘Clementinc’ × cultivars crossed with a tetraploid selection were observed to be polyembryonic. The 4x ‘Kawano Natsudaidai’. multiple embryos formed a small mass the the micropylar end. Plants regenerated in vitro from the embryos in polyembryonic seeds were triploid and showed identical The origin of these embryos was verified peroxidase banding patterns on acrylamide gels. These results indicate that the multiple by determining chromosome number and embryos found in the undeveloped seed from monoembryonic diploid × tetraploid analyzing the isozyme patterns of plants re- crosses are genetically identical and of zygotic origin. generated from eight polyembryonic seeds having two to 11 embryos. The embryos of Polyembryony is a common feature in Cit- to abortion of triploid embryos at various these seeds were cultured on Murashige and rus. Multiple embryos are produced from stages of embryogenesis (Esen and Soost, Tucker (1969) medium containing 500 mg nucellar cells. However, some cultivars have 1973). malt extract and 40 mg adenine/liter to in- been reported to produce two or more sexual The undeveloped seeds from these crosses duce plantlet formation. Chromosome counts embryos in one seed (Bacchi, 1943; Cam- were classified as monoembryonic, polyem- carried out by the root tip squash technique eron and Garber, 1968; Frost, 1926; Ozsan bryonic, or embryoless. The percentage of of Oiyama (1981) indicated that all plants and Cameron, 1963; Parlevliet and Cam- polyembryonic seeds was higher in the cross produced from any one polyembryonic seed eron, 1959; Ueno et al., 1967). Sexual poly- using ‘Miyauchi Iyokan’ as the seed parent were triploid (2n = 3x = 27) (Table 4). embryony may be due to multiple fission of (Table 2). The’ embryos in polyembryonic Multiple embryos formed in undeveloped the zygotic embryo or the presence of more seeds formed a small mass at the micropylar seeds are therefore of zygotic origin. Acryl- than one egg in an ovule (Bacchi, 1943). end (Fig. 2). These embryos were nearly amide gel electrophoretic analysis of per- Cameron and Garber (1968) presented cy- globular in shape but very dissimilar in size. oxidase isozymes according to the method tological evidence that supernumerary sexual The number of embryos differed greatly described by Kobayashi (1987) showed no embryos could occur by budding from the among seeds in each cross. The mean num- differences in banding patterns among the primary embryo. We describe polyembryony ber of embryos per seed was about half as triploid plants from the same polyembryonic in undeveloped seeds from mature fruit of great in ‘Clementine’ as in ‘Miyauchi Iyokan’ seed (Fig. 3). This similarity suggests that monoembryonic diploid cultivars crossed with (Table 3). the embryos in one undeveloped seed are a tetraploid and discuss its origin. Two monoembryonic diploid cultivars, Clementine mandarin (Citrus clementina Hort. ex Tanaka) and Miyarrchi Iyokan (C. iyo Hort. ex Tanaka) were hand-pollinated with tetra- ploid ‘Kawano Natsudaidai’ (C. natsudaidai Hayata) pollen. Hand pollination was carried out immediately following emasculation of flowers, and all the pollinated flowers were covered with bags. Seeds were extracted from mature fruit 7 months after pollination and were classified as well-developed or unde- veloped (Fig. .1). Embryo formation in un- developed seeds was observed under a binocular dissecting microscope. Most of the seeds produced by each seed parent in the cross with the tetraploid were undeveloped (Table 1). The frequency of normal seed development was greater for ‘Miyauchi Iyokan’ than for ‘Clementine’. The high frequency of undeveloped seeds from 2x-4x crosses, already reported by several ‘Seed parents were pollinated with 4x ‘Kawano Natsudaidai’, workers (Cameron and Soost, 1969; Esen and Soost, .1972, 1973; Oiyama et al., 1981; Tachikawa et al., 1961), has been attributed Table 3. Number of embryos in polyembryonic undeveloped seeds from monoembryonic diploid x tetraoloid Citrus crosses. z

Received for publication 4 Nov. 1989. Contribu- tion no. E-124 of the Fruit Tree Res. Sta. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regu- lations, this paper therefore must be hereby marked advertisement solely to indicate this fact. zSeed parents were pollinated with 4x ‘Kawano Natsudaidai’.

1276 HORTSCIENCE, VOL. 25(10), OCTOBER 1990 Table 4. Chromosome number of regenerated Cameron, J.W. and R.K. Soost. 1969. Characters Tree Res. Sta. D3: 1-7. plants from multiple embryos in undeveloped of new populations of Citrus polyploids, and Oiyama, L, N. Okudai, and T. Takahara. 1981. seeds from 2x ‘Clementine’ crossed with 4x the relation between tetraploidy in the pollen Ploidy levels of seedlings obtained from 2x × parent and hybrid progeny. Proc. 1st Intl. Citrus 4x crosses in citrus. Proc. Intl. Soc. Citriculture Symp. 1:199-205. 1:32-34. Esen, A. and R.K. Soost. 1972. Tetraploid proge- Ozsan, M. and J. W. Cameron. 1963. Artificial nies from 2x × 4x crosses of Citrus and their culture of small citrus embryos and evidence origin. J. Amer. Soc. Hort. Sci. 97:410-414. against nucellar embryony in highly zygotic va- Esen, A. and R.K. Soost. 1973. Seed develop- rieties. Proc. Amer. Soc. Hort. Sci. 87:210- ment in Citrus with special reference to 2x × 216. 4x crosses. Amer. J. Bot. 60:448-462. Parlevliet, J.E. and J.W. Cameron. 1959. Evi- Frost, H.B. 1926. Polyembryony, heterozygosis dence on the inheritance of nucellar embryony and chimeras in citrus. Hilgardia 1:365-402. in citrus. Proc. Amer. Soc. Hort. Sci. 74:252- Kobayashi, S. 1987. Uniformity of plants regen- 260. erated from orange (Citrus sinensis (L.) Osb.) Tachikawa, T., Y. Tanaka, and S. Hara. 1961. protoplasts. Theor. Applied Genet. 74:10-14. Investigations on the breeding of citrus trees. 1. Murashige, T. and D.P.H. Tucker. 1969. Growth Study on the breeding of triploid citrus vari- factor requirements of citrus tissue culture. Proc. eties. Bul. Shizuoka Citrus Expt. Sta. 4:33-44. 1st Intl. Citrus Symp. 3:1151-1161. Ueno, L, M. Iwamasa, and M. Nishiura. 1967. Oiyama, I. 1981. A technique for chromosome Embryo number of varieties of citrus and its observation in root tip cells of citrus. M. Fruit relatives. Bul. Hort. Res. Sta. B7:11-22.

HORTSCIENCE 25(10):1277-1279. 1990. Growth and Fruiting of Short- internode Main Dwarf. Normal- internode Parent, and Hybrid

Fig.2. Single (left) and multiple (right) embryos Dean E. Knavel and Robert L. Houtz formed in undeveloped seed from 2x ‘Clemen- Department of Horticulture and Landscape Architecture, University of tine’ × 4x ‘Kawano Natsudaidai’. Kentucky, Lexington, KY 40546 Additional index words. Cucumis melo, leaf area, leaf and stem dry weight, leaf area ratio, chlorophyll, ribulose-l,5-bisphosphate carboxylase/oxygenase, net assimilation rate, elemental concentration, yield Abstract. Plants of Main Dwarf, a short-internode mutant of the normal-internode ‘Mainstream’ muskmelon (Cucumis melo L.), have shorter internodes, fewer nodes, less total vine length, less total dry weight, smaller leaves, increased chlorophyll con- centrations, increased specific leaf dry weight, and increased ribulose-1, S-bisphosphate carboxylase/oxygenase (EC 4.1.1.39, rubisco) activity per unit leaf area than ‘Main- stream’ plants. Main Dwarf plants produce an equal number of fruit as ‘Mainstream’

plants but are only half their size. Many of the plant and fruit characteristics for F1 (Main Dwarf × ‘Mainstream’) are similar to those of ‘Mainstream’, except for greater

leaf chlorophyll and rubisco activity per unit leaf area. The F1 (’Mainstream’ x Main

Dwarf) produced fewer and lower weight fruit than its reciprocal, F1 (Main Dwarf x ‘Mainstream’).

Main Dwarf muskmelon is a short-inter- (Nerson et al., 1983). The prostrate spread- Fig. 3. Isoelectric focusing profiles of peroxi- node (SI) single gene recessive mutant (si- ing vine of Main Dwarf plants, which do not dase isozymes in triploid plants produced from 3) of the normal-internode (NI) cultivar have brittle and twisted internodes as re- one polyembryonic seed. Mainstream (Knavel, 1990). SI mutants pre- ported (Denna, 1962) for si-1 plants, maybe genetically identical and derived from a sin- viously reported were of the si-1 (Davis et of value in the development of SI genotypes gle fertilized egg cell. al., 1976; Knavel, 1988a; Zink, 1978, 1979) with the potential of higher yields on per unit The present study demonstrates that sex- and of the si-2 gene type (Paris et al., 1984). area than now used for NI cultivars. The ual polyembryony of citrus occurs at the trip- Main Dwarf plants have more-branching, less- objective of this study was to document veg- loid level. This sexual polyembryony seems compact, and more-open vines and have etative growth, fruiting, and leaf character- to be a specific phenomenon related to ab- smaller leaves than other SI genotypes such istics of SI Main Dwarf plants and compare

normal development of triploid embryos in as Ky-P7 (Knavel, 1988a) and Persia-202 these characteristics with those of the NI par- monoernbryonic diploid × tetraploid crosses. plants that exhibit an upright growth habit ent ‘Mainstream’ and their reciprocal hy- brids. Literature Cited Received for publication 24 Aug. 1989. Published Each year (1985, 1986, 1987) seed were Bacchi, O. 1943. Cytological observations in Cit- as 89-10-178 Agricultural Experiment Station Ar- sown in plastic pots (0.1 liter) containing a rus. 111. Megasporogenesis, fertilization and ticle. The cost of publishing this paper was de- commercial peat moss and perlite mixture. polyembryony. Bot. Gaz. 105:221-225. frayed in part by the payment of page charges. The plants were grown for 30 days in a Cameron, J.W. and M.J. Garber. 1968. Identical- Under postal regulations, this paper therefore must plastic greenhouse (from 10 to 15 Apr. to twin hybrids of Citrus x Poncirus from strictly be hereby marked advertisement solely to indicate mid-May) before being transplanted to the sexual seed parents. Amer. J. Bot. 55:199–205. this fact. field onto rows spaced 1.8 m apart, center

HORTSCIENCE, VOL. 25(10), OCTOBER 1990 1277