Preliminary observation on a spontaneous tricotyledonous mutant in sunflower Jinguo Hu 1, Jerry F. Miller 1, Junfang Chen 2, Brady A. Vick 1 1 USDA, Agricultural Research Service, Northern Crop Science Laboratory, Fargo, ND 58105 2 Department of Plant Sciences, North Dakota State University, Fargo, ND 58105 Abstract This report documents the tricotyledonous phenotype in sunflower. The mutant was found in a BC 3 F 2 population developed for incorporating sulfonylurea (SU) herbicide resistance into cultivated sunflower. Progeny tests were carried out for three consecutive generations to study the inheritance of this anomalous characteristic under both greenhouse and field conditions during the past two years. It was interesting to observe that tricotyledonous seedlings also produced true leaves in sets of three at the first few nodes. The frequency of tricotyledonous phenotype increased from approximately 2% in the F 2 generation to about 50% in the F 5 generation (34 of 71). This suggested that this peculiar phenotype might result from recessive genes of low penetrance. Additional studies are needed to elucidate its genetic base. Introduction Tricotyledonous seedlings (seedlings with three cotyledons, or tricot) occur sporadically in nurseries of dicotyledonous plant species, including sunflower. Researchers have documented this phenomenon in many plant species such as tomato (Reynard, 1952; Haskell, 1962; and Kerr, 1985), mustards (Holtorp, 1944) and snapdragon (Harrison, 1964). In most cases, the tricotyledonous phenotypes did not produce pure tricotyledonous progeny and the inheritance seemed complex. However, Rai and Kumar (2001) reported a tricotyledonous mutant in Catharanthus roseus controlled by a single recessive gene. Molecular studies have shown that a few mutated genes could produce the tricotyledonous trait in the model plant Arabidopsis (Azumi et al., 2002; Vernon et al., 2001; Conway and Poethig, 1997). In sunflower the phenotype of multiple cotyledons was reported by Skaloud and Kovacik (1978). In this paper we report the results of observations on the progenies of a tricotyledonous mutant in sunflower. Materials and methods Origin of the mutant Seven seedlings possessing three cotyledons were found in the BC 3 F 2 population derived from the cross HA 434//HA 406//HA 89/SU Resistant wild H. annuus (Fig. 1). The pedigree of this cross is as following: Plants of a wild H. annuus population collected in Kansas were screened for resistance to the sulfonylurea herbicide, chlorsulfuron [2-chloro-N-[(4-methoxy-6- methyl-1,3,5-triazin-2-yl) aminocarbonyl] benzenesulfonamide]. Resistant plants were grown in the greenhouse at Kansas State University during the fall of 1998, and pollen was collected. The pollen was transferred to the USDA-ARS Sunflower Genetics Project, Fargo, ND, in the spring of 1999, and was used to pollinate HA 89. HA 89 (PI 599773) is an oilseed maintainer line
released by the USDA-ARS and the Texas Agricultural Experiment Station in 1971. True F 1 hybrids were obtained using an embryo rescue procedure. Pollen from SU resistant F 2 plants was used to pollinate HA 406. HA 406 (PI 597370) was released by the USDA-ARS and the North Dakota Agricultural Experiment Station in 1993 (Miller and Gulya, 1997). Pollen of the SU resistant plants was used to pollinate HA 434. HA 434 is a high oleic oilseed line released by the USDA-ARS, Fargo, ND, and the North Dakota Agricultural Experiment Station, Fargo, ND, in 2001 (Miller et al., 2004). Since there were no tricotyledonous seedlings found in the HA 89 x SU Resistant mutant HA 406 x F 1 HA 434 x F 1 F 1 F 2 Figure 1. Pedigree of the tricotyledonous mutant. Eight F 2 families with a total of 334 seedlings were grown for SU resistance test in the greenhouse. Seven seedlings possessing three cotyledons were found and transplanted into one gallon pot and selfpollinated to carry out the progeny test for the three cotyledon characteristics. self-pollinated progenies of HA 89, HA 406 and HA 434, the tricotyledonous phenotype could have originated from the wild sunflower or from the interaction of genes from both cultivated and wild sunflower accessions. Progeny tests The mutant plants with three cotyledons were self-pollinated to produce seeds for progeny testing in three consecutive generations. Observations were made under both greenhouse and field conditions from 2002 to 2004. Results and Discussion Characteristics of the tricotyledonous plants Normal sunflower seedlings have two cotyledons (Fig. 2a). The mutant seedlings have three cotyledons (Fig. 2b). No significant detrimental effect on growth and development was
observed with the mutant plants since all plants produced self-pollinated progeny. In the progeny, seedlings with one and four cotyledons were observed at very low frequency. In seedlings with two cotyledons there are two true leaves per node (Fig. 2c). Most of the tricotyledonous plants produced three true leaves at each node in the first six to seven internodes (Fig. 2d). The internodal length between the normal and the mutant was about the same. The Figure 2. Normal (A and C) and tricotyledonous (B and D) seedlings of different ages. Top: one week old in the greenhouse; Bottom: about twenty days after planting in the field. size and the shape of the leaves did not differ. The height of the tricot plant was nearly the same as that of the dicotyledonous plants. Frequency of tricotyledony in three generations In 2002, seven tricotyledonous seedlings were found in the BC 3 F 2 population of 334 seedlings, a frequency of approximately 2%. These seven plants were transplanted into the greenhouse and five plants survived to maturity. The observed tricotyledonous frequencies among the five F 3 families ranged from 3 to 31% with an average of 18.5% (157 tricot in 755
seedlings counted) in the field in 2003. We bagged at random twelve plants to produce F 4 progenies. Four F 4 families selected from these 12 for the higher frequency of tricotyledonous seedlings were planted in the field in 2004. The observed frequencies among the four F 4 families ranged from 15 to 55% with an average of 35.5% (122 tricot in 383 seedlings counted). The same test was repeated for four F 5 families and the frequency ranged from 15% to 45% with an average of 24.1% (72 tricot in 399 seedlings counted). These results suggested that this anomalous characteristics is heritable. However, since the tricotyledony could not be fixed after self-pollination for three consecutive generations and no significant gain was observed from F 4 to F 5 generation, tricotyledony in this mutant seems to be derived from recessive genes of low penetrance. This mutant did not segregate in the 13:3 ratio of dominant inhibition as mentioned by Skaloud and Kovacik (1978). Additional studies are needed to elucidate its genetic base. Potential applications Cotyledons are the major component of the sunflower seeds, which are harvested for oil or confection uses. The changing in cotyledon number in the embryo could be related to both yield and quality. The extra cotyledon could help the seedlings become established after germination since the cotyledons function is to provide nutrients such as lipids, proteins, and carbohydrates that the growing plant needs before it reaches the point where it can make its own supply through photosynthesis. Another interesting aspect is that the mutant has three true leaves per node, therefore, the seedling will have larger leaf area for photosynthesis. The development of a true breeding tricotyledonous line would help address important questions related to sunflower production. Reference Azumi, Y., D. Liu, D. Zhao, W. Li, G. Wang, Y. Hu, and H. Ma. 2002. Homolog interaction during meiotic prophase I in Arabidopsis requires the SOLO DANCERS gene encoding a novel cyclin-like protein. EMBO J. 21:3081-95. Conner, J. K., and A. A. Agrawal. 2004. Mechanisms of constraints: the contributions of selection and genetic variance to the maintenance of cotyledon number in wild radish. J. Evol. Biol. 18: 238-242. Conway, L. J., and R. S. Poethig. 1997. Mutations of Arabidopsis thaliana that transform leaves into cotyledons. Proc. Natl. Acad. Sci. USA 94:10209-10214. Holtorp, H. E. 1944. Tricotyledony. Nature 153:13 14. Kerr, E.A. 1985. Virescent-3 (v-3), a new mutant possibly on Chromosome 4. Tomato Genetics Cooperative Newsletter 35:6. http://gcrec.ifas.ufl.edu/tgc/newsletters/vol35/v35p6a.html. Miller, J. F., and K. Al-Khatib. 2004. Registration of two oilseed sunflower genetic stocks, SURES-1 and SURES-2 resistant to tribenuron herbicide. Crop Sci. 44:1037-1038. Miller, J. F., and T. J. Gulya. 1997. Registration of eight maintainer (HA 393, HA 394 and HA 402 to HA 407) and seven restorer (RHA 395 to RHA 401) sunflower germplasm lines. Crop Sci. 37:1988-1989.
Miller, J. F., T. J. Gulya and B. A. Vick. 2004. Registration of two maintainer (HA 434 and HA 435) and three restorer (RHA 436 to RHA 438) high oleic oilseed sunflower germplasms. Crop Sci. 44:1034-1035. Rai, S. P. and S. Kumar. 2001. A tricotyledonous seedling mutant with Mendelian inheritance in periwinkle Catharanthus roseus. J. Med. Arom. Plant Sci. 22:267-268. Reynard, G. B. 1952. Inheritance of polycotyledony in tomato. Tomato Genetics Cooperative Newsletter 2:2. http://gcrec.ifas.ufl.edu/tgc/newsletters/vol2/v2p2.html. Skaloud, V. and A. Kovacik. 1978. Survey on inheritance of sunflower characters which are conditioned by a small number of genes. P.490-496. In Proc. 8 th Int. Sunflower Conf., Minneapolis, MN. July 23-27, 1978. Int. Sunflower Assoc. Paris, France. Vernon, D. M., M. J. Hannon, M. Le, and N. R. Forsthoefel. 2001. An expanded role for the TWN1 gene in embryogenesis: defects in cotyledon pattern and morphology in the twn1 mutant of Arabidopsis (Brassicaceae). Am. J. Bot. 88:570-582.