FACTORS INFLUENCING GERMINATION AND GROWTH OF ISOLATED EMBRYOS OF PINUS HELDREICHII

The effects of nutritional, hormonal, and physical factors on the germination and growth of isolated mature zygotic embryos of Pinus heldreichii were studied under in vitro conditions. The optimum medium for embryo germination and adequate conversion into seedlings was basal Gresshoff and Doy (1GD) medium. All tested carbohydrates at the applied concentrations stimulated embryo growth, but only 3% sucrose increased embryo germination compared to the control, while maltose had an inhibitory effect. Among the applied plant growth regulators, only gibberelic acid (GA3) had a few benefits on the development of isolated embryos into plantlets, while the others had a negative effect. Embryos cultured on an agar-solidified medium grew better and the germination percentage was higher than in a liquid medium. Light treatment did not affect embryo germination, but generally stimulated seedling growth.


INTRODUCTION
Pinus heldreichii (Bosnian pine) is a Tertiary relic species endemic on high mountains in the Balkans and Southern Italy.It occurs on steep and dry limestone slopes, most often in pure stands (V i d a k o v i ć , 1982).The tree is ornamental, up to 30 m high, with pyramidal habit.Although the tree grows slowly, it could be important for afforestation, as it is well adapted to environmental stresses such as low temperature and extreme drought (J o v a n o v i ć , 1971).In natural stands, trees begin to produce cones at the age of about 40.Seeds are dormant and need chilling treatment of about 6 weeks to germinate (S t i l i n o v i ć , 1985).Abundance of seed production varies greatly, not only from year to year, but also within different trees and cones (Ð o r đ e v a et al., 1972; M i č e v, 1972).Pinus heldreichii is usually propagated by seeds, since the rooting of cuttings has been only partially successful.However, variable seed production may complicate operational reforestation and tree improvement efforts.In vitro culture can therefore be used as a significant supplement to conventional methods of vegetative propagation of this pine.
We previously reported plant regeneration of P. heldreichii through adventitious buds (S t o j i č i ć et al., 1999) and axillary buds (S t o j i č i ć and B u d i m i r , 2004).We also demonstrated induction of somatic embryogenesis in culture of this pine (S t o j i č i ć et al., 2007).According to the obtained results, micropropagation and somatic embryogenesis have a potential use in the propagation of P. heldreichii, but further improvement of the methods is required.
The aim of this study was to investigate the possibility of achieving synchronous and rapid plant production from zygotic embryos using tissue culture methods.To that end, we investigated the effects of nutrients, hormones, and physical factors on germination and growth of isolated mature zygotic embryos.

Seed sterilization and culture of isolated embryos
Cones of P. heldreichii were collected from open pollinated trees in a natural stand located on Lovćen Mountain (Montenegro).Before the experiments, seeds were removed from cones, washed for 24 h under running tap water, surface-disinfected in 20% sodium hypochlorite for 30 min, and rinsed three times with sterile distilled water.Isolated mature zygotic embryos were then placed horizontally on different variations of GD (G r e s s h o f f and D o y, 1972) culture medium as modified by S o m m e r et al. (1975).If not mentioned otherwise, the medium used was supplemented with 3% sucrose and solidified with 0.7% agar (Torlak, Belgrade).The pH of the media was adjusted to 5.7 prior to autoclaving for 25 min at 115ºC.Unless stated differently, cultures were maintained at 25 ± 2°C under conditions of a 16 h/8 h photoperiod.

Variations of media
Medium strength -the effects of five different medium strengths were tested: 0; 0.25; 0.5; 1.0; or 2.0 times those of GD.The other components in all five media were the same as in GD medium.
Effects of carbohydrates -to determine the influence of different carbon sources, GD medium was supplemented with one of four carbohydrates (sucrose, glucose, maltose, or fructose), each at six different concentrations (0; 1; 2; 3; 4; or 5%).
Effect of enzymatic casein hydrolyzate (CH) -as a source of organic nitrogen, CH was tested at the following concentrations 0; 125; 250; 375; or 500 mg l -1 .
These media were also supplemented with CH at 500 mg l -1 .
Comparison of agar-gelled and liquid media -two different treatments were compared: 0.7% agar-gelled medium and liquid medium.The basal 1GD medium supplemented with CH at 500 mg l -1 was used.

Effect of light
Three different treatments were compared in this study: continuous light; 16-h light photoperiods; and continuous darkness.

Data collection and analysis
For each treatment, a total of 36 embryos (six per Petri dish) were used, divided into two replicates.Embryos were scored as germinated if they exhibited root elongation.The dry weight of plantlets was recorded after drying at 70ºC for 24 h.Germination percentages were arcsine transformed before analysis.Data collected from experiments were calculated and differences were tested for significance using ANOVA and Duncan's multiple range test at a significance level of p ≤ 0.05.

RESULTS AND DISCUSSION
The germination percentage of isolated embryos after four weeks was only slightly affected by medium strength, in spite of significant differences observed during the first two weeks of embryo cultivation (Fig. 1).On the medium without salts and vitamins, radicle elongation was poor, cotyledons did not open, and embryo germination was delayed (Fig. 2).A similar delay was also observed on double-strength (2GD) medium, probably due to high osmotic stress.Supplementation of the medium with salts and vitamins promoted elongation of the radicle, followed by elongation of the hypocotyl and expansion of the cotyledons.The highest frequency of embryo germination was obtained on basal 1GD medium, where an average of 80% of embryos germinated after one week in culture (Fig. 1).Basal 1GD medium stimulated embryo growth, so that at day 28 seedlings were two times longer (42.8 mm) than on control 0GD medium (22.7 mm).Root growth was best on 1GD medium (Table 1) and these roots were branched, with prominent root hairs.The 1GD variant also promoted embryo transformation into seedlings and shoot development (Fig. 3).L i n and L e u n g ( 2002 Among carbohydrates tested as a carbon source, sucrose was superior in promoting Pinus heldreichii embryo germination compared to glucose, fructose, and maltose (Table 2).All sucrose concentrations (1-5%) slightly increased germination frequency com-pared to the control.In the presence of 5% sucrose, germination was delayed, so that after one week in culture about 60% of embryos germinated, while at the end of the fourth week germination increased to nearly 80%.Glucose and fructose had no effect on germination, while maltose had an inhibitory effect (Table 2).Similar results were obtained in Pinus radiata, where sucrose, glucose, and fructose could serve as carbon sources for isolated embryos cultured in vitro, while maltose was found to be inferior to these sugars in promoting germination and embryo growth (L i n and L e u n g , 2002).However, unlike isolated mature embryos of P. radiata, which could not germinate on a medium without any carbohydrates, P. heldreichii embryo germination in the absence of a carbon source was relatively high (72%).Growth of embryo organs and whole seedlings was stimulated by the presence of the carbohydrates in the medium irrespective of sugar type (Table 2).The addition of organic nitrogen in the form of enzymatic casein hydrolyzate in the medium did not increase germination percentage, but promoted the growth of hypocotyl and cotyledons at concentrations from 250 to 500 mg l -1 (data not shown).
None of the plant growth regulators tested affected isolated embryo germination at the applied concentrations (Table 3).However, in the presence of GA 3 , germination was synchronous and most embryos germinated after one week of treatment.In addition, GA 3 at lower concentrations slightly promoted hypocotyl elongation, while the highest seedling dry weight was obtained at 1.15 µM GA 3 (Table 3).Gibberellic acid was also shown to increase germination percentages and promote embryo transformation into plants in P. radiata and some other woody species, like coconut (L i n and L e u n g , 2002; P e a c h et al., 2007).Cytokinins and auxins were generally inhibitory for early seedling growth, especially cotyledon and root growth.The growth of isolated zygotic embryos was significantly reduced  in the presence of either BA or KIN, independently of the concentrations applied (Table 3).However, these cytokinins did not affect shoot development, although they influenced the formation of callus tissue along the hypocotyl.In the presence of BA at 0.44 µM and KIN at 0.23 and 0.46 µM, adventitious buds were formed along the cotyledons.The effects of culture media and BA concentrations on adventitious bud induction and elongation were previously reported by S t o j i č i ć et al. (1999).Isolated embryos grown in the presence of auxins (NAA and IBA) showed abnormalities, viz., long and crinkled cotyledons, and short and swollen hypocotyls on which callus tissue was formed.Naphthaleneacetic acid suppressed the growth of isolated embryos, especially root growth.Although inhibition of root growth by low concentrations of auxin is a wellknown phenomenon, in Pinus pinea zygotic embryos NAA was found to be necessary for root growth (D i a m a n t o g l o u et al., 1990).
In contrast to germination, which was indepen-dent of light (Table 4), the growth of germinated embryos was influenced significantly by different light treatments, among which a 16-h photoperiod appeared to be the best for root and cotyledon growth (Table 4).Seedling dry weight increased significantly when grown in light, compared to continuous darkness.The frequency of embryo germination was higher on agar-solidified medium than in liquid medium (Table 4).In addition, embryos cultured on agar-solidified medium had significantly better growth (Table 4) and developed into healthy-looking plantlets.
The results obtained in this study showed that isolated zygotic embryos germinated readily under in vitro conditions after only one week in culture.Although embryos could germinate and grow on a medium without salts, vitamins, and carbohydrates, the highest germination percentage and the best morphological characteristics of the seedlings were obtained on basal 1GD medium supplemented with 3% sucrose.Exogenous growth regulators are not Table 4. Effect of physical factors on the germination and growth of isolated embryos of Pinus heldreichii.All data were obtained at day 28.Means in the column followed by different letters are different according to Duncan's multiple range test (p ≤ 0.05).
) obtained similar results in Pinus radiata zygotic embryo culture, where only a few embryos survived on a medium without salts and vitamins, while the highest germination rate and maximum growth were achieved on half-strength medium consisting of Quoirin and Le Poivre salts (v o n A r n o l d and E r i k s s o n , 1981) and S c h e n k and H i l d e b r a n d t (1972) vitamins.

Fig. 1 .
Fig. 1.Effect of medium strength on mean germination percentage of isolated embryos of Pinus heldreichii.Error bars indicate the standard error of the mean.

Table 1 .
Effect of medium strength on the growth of isolated embryos of Pinus heldreichii.All data were obtained at day 28.Means in the column followed by different letters are different according to Duncan's multiple range test (p ≤ 0.05).

Table 2 .
Effect of carbon source on germination and growth of isolated embryos of Pinus heldreichii.All data were obtained at day 28.Means in the column followed by different letters are different according to Duncan's multiple range test (p ≤ 0.05).

Table 3 .
Effect of growth regulators on germination and growth of isolated embryos of Pinus heldreichii.All data were obtained at day 28.Means in the column followed by different letters of each growth regulator are different according to Duncan's multiple range test (p ≤ 0.05).