Breaking the interspecific-hybrid sterility in Iris In vitro chromosome doubling and subsequent flowcytometric analysis Iribov Evelien Huis in ‘t Veld*, Eloy Boon* and Christoph Boon** * Iribov - Westeinde 149a - 1601 BM - Enkhuizen, The Netherlands ** Breeding group Juno - P.O.Box 43 - 1600 AA Enkhuizen, The Netherlands Introduction Materials and methods Nuclear DNA amount of the plants was calculated by using nuclei of Allium cepa as internal standard Most major Iris cultivars derived from crosses For obtaining in vitro material, the tissue culture which has a nuclear DNA amount of 33,5 pg. between I. xiphium (2n=34) and I. tingitana method was used as described by Van der Linde et (2n=28) sterile hybrids (2n=31) and therefore al. (1990). Iris bulb scale explants were used for cannot be used for further breeding. in vitro initiation. Obtained plants were multiplied by using the longitudinal split method. Six months There is one case known in which fertility was before planting bulb formation was induced. restored spontaneously by natural occurring poly- ploidization. Among the offspring of this plant Colchicine treatment was given to explants after are some important Iris cultivars (e.g. Apollo, multiplication. Several colchicine concentrations Telstar, Saturnus). were used. In this poster work is presented in which in tetra- Plants derived from explants surviving colchicine ploid irises are produced. This poster presents an treatment were analyzed with a Partec CA-II flow- example of in vitro polyploidization of Iris by cytometer according to De Laat (1986) Nuclei threating young plantlets with colchicine. This were stained with a High Resolution Kit (Partec). procedure is based on the method described by Eikelboom & Van der Linde (1992). Figure 1 Dutch Iris bulb Nuclei / Results channel Table 1 Nuclear DNA content of Iris 200 cultivars and of interspecific hybrids Colchicine reduced regeneration from explants Iris tingitana considerably. Although depending on the cultivar, (2x = 28) cultivar Expected Measured Iris xiphium some specific treatments developed by Iribov 150 (2x = 34) DNA DNA achieved reasonable shoot formation. amount amount Tetraploids were easily detected by flowcytometry 100 Allium cepa 33,5 (Figure 2). In the surviving plants the percentage Iris xiphium 11,4 of tetraploid plants varied in a range of 3-30% Iris tingitana 14,6 with an average of 10%. Fortunately in all culti- 50 Allium cepa vars tested, tetraploid plants were found. I. xiph. x I. ting (= I. hollandica) (11,4 + 14,6) / 2 = ) 13,0 13,1 Flowering tetraploid Iris plants, proved to be 0 I. hollandica (4n) fertile, although seed production was lower com- Channel number = relative DNA amount pared to diploid I. xiphium or I. tingitana plants. ( 2 * 13,0 = ) 26,0 26,3 Figure 3. The nuclear DNA amount of Iris xiphium, Iris tingitana I. hollandica (4n) x I. xiphium and of the internal standard Allium cepa. ( 26,0 + 11,4 ) / 2 = ) 18,7 18,9 I. hollandica (4n) x I. tingitana Nuclei/ 1200 ( 26,0 + 14,6 ) / 2 = ) 20,3 20,4 channel Differences in nuclear DNA amounts were 1000 Diploid plant 800 observed between the different diploid Iris species 600 (figure 3). I. tingitana showed to have a higher cv. Apollo 18,7 18,8 400 0,1,02468 200 DNA amount compared to I. xiphium. The inter- 0 0,1,02468 specific hybrid had an intermediate DNA amount Nuclei/ channel 600 (table 1). 500 Tetraploid plant 400 300 Assuming that the nuclear DNA amount should 200 References 100 be intermediair between the parental values, the 0 expected DNA amount of other combinations was Channel number = relative DNA amount calculated. Analyses of these plants confirmed - Bennet, M.D. & J.B.Smith,1976. Phil trans. R. the expected value (Table 1). In addition the Soc. London Ser B. 227: 28-274. natural polyploid cultivar Apollo contained a - Eikelboon, W. & P.C.G. van der Linde, 1992. Figure 2. The nuclear DNA amount of a diploid Iris and a tetraploid similar DNA amount (18,8 pg) according to the Bloembollencultuur 7: 22. Iris. calculated amount of (I. xiphium x I. tingitana) x - Laat, L. de et al, 1987. Plant Breeding 99, 303- (I. xiphium), the assumed parents. 307. - Linde. P. van der et al, 1990. Bloembollencultuur 10:29-31.