IMPROVEMENT OF ANDROGENIC EMBRYO GERMINATION AFTER COOLING TREATMENT OF AESCULUS

Androgenic embryos from anther (R ado j e vic 1978, R ado j e vic 1991) and microspore culture (R ado j e vic et al. 1998) showed rapid differentiation, asynchronous development and low germination. Cool treatment (6°C) is known to improve germination of somatic embryos of horse chestnut (P r 0 fum

The samples were collected from a llO-year-old A. hippocastanum tree growing in the Jevremovac Botanical Garden of the Belgrade University.Sterilization, isolation and cultivation of anthers were performed according to a previously elaborated procedure (R ado j e vic 1978, R ado j e vic et al. 1998).Basal (MS) medium containing M u r ash i g e and S k 0 0 g (1962) mineral solutions and 2% sucrose was supplemented with lmg L•lj: pantothenic acid 10.0, nicotinic acid 5.0, vitamin B 1 2.0, adenine sulphate 2.0, myo-inositol 100 and casein-hydrolysate 200.Seven anthers from each bud were cultured in tubes containing MS l solid medium with 2,4-D and Kin (1.0 mg L-l, each), while the uninuclear microspores were cultured in MS2 liquid medium with the same content of both hormones (C a lie et al. 2003).To obtain suspension culture 100 transverse-cut anthers with uninuclear microspores per Erlenmeyer flask in 100 cm 3 MS2 medium were used.Liquid cultures were filtered (200 1Jrn) to obtain a suspension.Microspore suspension was subcultured every 4 weeks and refreshed with MS 2 medium.After 2 months, microspore suspension was plated by B erg man n technique (1960) on a MS solid medium with reduced concentration of 2,4-D (MS3 = MS + 0.01 mg L'! 2,4-D + 1.0 mg L-l Kin ).Embryo development and multiplication of androgenic embryos from anther and suspension culture proceeded on MS 3 medium.After the multiplication, embryos were cultured on a germination medium (MS4 =MS + Glu 400 mg L-l).
All media were sterilized by autoclaving at 0.9 x lOs KPa and 114°C for 25 min.Suspension cultures were grown on horizontal shaker (85 rpm) at 25 ± 1 °c for 4 weeks in the dark, while the other cultures were grown under identical temperature and a 16-h photoperiod with irradiance of 33 -45 umol m• 2s-1 produced by cool white fluorescent tubes.The experiment was repeated 4 times.
Embryos with cotyledons were grown on MS4 medium and in cool storage at 6 °c during 4 and 6 months.Adventive embryos were normally formed on roots.Embryos that germinated after chilling were transferred to the same medium (MS 4) and grown for one month at 25 ± 1 °c and 16 h light/8 h dark photoperiod.
The least significant difference (LSD) test showed the increase in root and plantlet length to be significantly lower in microspore suspension than in anther culture (Table 1).Cooling (6°C) over a period of 6 months had a favourable effect on elongation of roots and total growth of plantlets (Table 1).Our results are in agreement with the findings of Pro fu m 0 et al. (1991) on the chilling of A. hippocastsanum somatic embryos.This method of chilling of androgenic embryos will be applicable in resolving the problem of dormancy.

Table 1 .
Influence of cooling (6°C), on an increase of roots and plantlets length, derived from anther culture and microspore suspension of A. hippocastanum L., after 4 and 6 months.In each column, the values with different letters are significantly different at the 0.05 probability level according to protected LSDtest. *