THE INFLUENCE OF SUGAR SOURCE IN INDUCTION CULTURAL MEDIA ON THE EFFECTIVENESS OF CALLUS FORMATION..

Double haploids are important for durum wheat breeding. Anther culture method for obtaining double haploid is one of the most popular. Effectiveness of this method de­ pends on different factors such as cultural condition and plant genotype. It is important to investigate the impact of cultural condition on the level of callus induction and plant regeneration. Sugar source in cultural media affects a process of androgenesis in vitro. Ability of four winter and four spring durum wheat hybrids F2 to androgenesis in vitro was studied. Two induction media with different sugar source were compared. C17M media contained maltose in 90 g/l concentration. C17n media contained sucrose in 60 g/l concentration and glucose in 17.5 g/l concentration. It was estimated that the level of callus induction and albino plant regeneration were higher on the media with maltose for all investigated genotypes. The level of green plants regeneration was low. Green plants were obtained only for three genotypes. Three green double haploids of T12 genotype obtained from new formations on C17M media, were successfully culti­ vated in soil and produced seeds. Analysis of variances (ANOVA) was used for statistical analysis. The percentage of total variance accounted for each factor was calculated. It was established that plant genotype had the most significant influence on callus induc­ tion. Interaction between factor of genotype and induction cultural media had the biggest percentage of total variance accounted for albino and green plants regeneration.


INTRODUCTION
Double haploid is an efficient biotechnological method in wheat breeding which can significantly accelerate the process of production of new varieties. In vitro anther culture is one of the most important methods of haploid plants obtaining. Presently we have only limited knowledge about androgenesis in wheat durum. It was reported that this cereal is recalcitrant regarding anther culture because of its low ability to regenerate green plants [1,9]. Several factors which influenced anther culture have been studied, including geno type of the explants [1] and culture conditions [17].
Most of the induction cultural media contain high sugar concentration (6-10%). Nutritional requirements for androgenesis and formed embryos growth are diffe rent [19]. For many plant species maltose has been known as a better carbohydrate source in comparison to sucrose for androgenesis. The use of maltose instead of su crose as a carbohydrate source in culture medium led to a substantial stimulation of microspore embryogenesis, plant regeneration, and green plantlet formation [6,10,11].
The effects of maltose have been documented in several tissue culture systems, but the reason for their superiority to sucrose is not known. It was demonstrated that maltose improves the osmotic stability of culture medium compared to sucrose [4]. It has also been reported that maltose stimulates embryogenesis at low concentra tions [14]. It was estimated that utilization of maltose as sugar source lasted for a longer period due to a slower rate of hydrolysis compared to sucrose [8]. Besides, it is as sumed that fructose, which is a side product of sucrose hydrolysis, inhibits androgene sis in vitro [5]. Maltose may be more effective source of sugar due to the fact that the rate of hydrolysis is close to the rate of glucose uptake during embryos germination [12].
Effect of sugar agents on durum culture of wheat anther were tested in several in vestigations [9,16]. The anthers were cultured on media with different carbon sources: sucrose and maltose. Supplements in culture medium significantly affected calli induc tion and plant regeneration. The number of calli and plants on the medium with maltose were significantly higher than the medium with sucrose. The green plant regeneration was effected by the initiation medium as well as genotypes. Maximum response was found on the medium with maltose.
The aim of this investigation was to determine the impact of sugar source in the induction cultural media on the effectiveness of calli production and plant regeneration in durum culture of wheat anther in vitro.
Two induction cultural media with different sugar source were compared. The influ ence of plant genotype and cultural media on callus induction and plant regeneration in vitro was estimated.

MATERIALS AND METHODS
Four spring and four winter wheat durum hybrids F2 were used as anther donors (Table 1). For spike pretreatment low positive temperatures (+3-+5 °C) in water for 7 days were used [15]. Spikes were sterilized with calcium hypochlorite solution using the me thod [3]. The experiment was set up with two induction media: C17n nd C17M. Induction media were identical except the source of sugar. C17n media contained 6 % of sucrose and 1.75 % of glucose and C17M media contained 9 % of maltose. Both media contained 2 mg/l 2,4D and 0.5 mg/l kinetin [18]. Anthers were incubated in dark at +24 °C [2].
After 15-25 days of cultivation new formations were transferred to regeneration media MS cultural media with BAP (1 mg/l) and IAA (1 mg/l), proline (200 mg/l) and glutamine (200 mg/l). New formations were incubated in dark. Callus with regenerative zones or embryos were transferred to Ѕ MS media without growth regulators [7] and in a growth room at 25 °C with 16 h light /8 h dark photoperiod [2].
The level of induction and regeneration was calculated as a callus and albino and green regenerated plants percentage upon total number of callus and confidence inter val for each experimental variant. Analysis of variance (ANOVA) was used for statistical analysis [13].

RESULTS AND DISCUSSION
Level of induction of all tested genotypes was higher on C17M media (Fig.1). Albino plant regeneration of spring hybrids was observed only for callus on C17M media. Le vel of winter hybrids albino plant regeneration was higher for callus on C17M media (Fig. 2).  T1  T8  T12  T15  T42  T43  T44  T45   C17n   C17M Level of green plant regeneration was low. On C17M media it was 0.51±0.51 % for T12 and 0.26±0.19 % for T43. On C17n media it was 0.77±0.54 % for T32. Three green plants of T12 were successfully adopted in soil and cultivated in growth chamber. Plants were double haploids and produced seeds. Green plants of T32 and T43 were defective and could not be adopted in soil. In general, the media with maltose was more effective for callus induction and plant regeneration for most of studied plant genotypes.    Genotype   T1  T8  T12  T15  T42  T43  T44  T45  ISSN  Twofactor analysis of variance with factors of plant genotype and induction cul tural media was used to check the impact of sugar sources and plant genotype on the level of callus induction and albino and green plant regeneration (Table 2). Two fac tors and its interaction affected significantly both the level of callus induction and plant regeneration. The highest percent of total variance for callus induction was 55.89 % accounted for genotype factor. The highest percent of total variance for albino and green plant regeneration was 35.62 % and 39.28 %, correspondingly. It was accounted for interaction between two factors. Percent of total variance for factor of induction cul tural media was 16.92 % for callus induction, 8.13 % for albino plant regeneration and 8.56 % for green plant regeneration. Since induction media were different only with sugar source, it can be concluded that maltose had a significant influence on in vitro callus induction and plant regeneration.

The influence of sugar source and genotype on the level of callus induction and
plant regeneration in wheat anther durum culture in vitro was estimated. It was found that sugar source had a significant influence on wheat anther durum cul ture effectiveness. 2. Maltose is shown to be more effective sugar source comparing with sucrose. It was established that the level of callus induction and plant regeneration was higher on the media with maltose comparing with media with sucrose and glu cose. 3. Three fertile double haploid plants of T12 genotype were obtained.