THE EFFECT OF LOW INITIAL MEDIUM pH ON IN VITRO WHITE POPLAR GROWTH

The effect of low initial medium pH on shoot and root development of five white poplar (Populus alba L.) genotypes was tested. The shoot height, fresh mass of shoots per jar, dry mass of shoots per jar, number of roots, as well as the length of the longest root were measured and final pH of the media determined, after 35 days of culture in vitro. Three initial pH values of the medium were tested: 3.0, 4.0 and 5.5 as control. Agar solidification at pH 3.0 was not achieved after sterilization in autoclave, but it was successful after sterilizing in a microwave oven. The obtained results indicate that the tested genotypes are able to significantly influence the changes of media pH during culture. The effect of differences among the examined media was significant for biomass accumulation and final media pH. Generally, significantly higher values of fresh and dry shoot mass, shoot height and the longest root length were recorded on a medium with initial pH 3.0 then on a standard medium with pH 5.5.The implications of the obtained results for the improvement of in vitro propagation of white poplars are discussed.


INTRODUCTION
White poplar (Populus alba L.) is a tree species that is widely spread throughout Europe, eastern Asia and northern Africa.However, in spite of its high adaptability, it is considered to be a threatened species and an indicator of biodiversity (K o v a c e v i c et al., 2010a).Beside its use for wood and biomass production, this species has a wide implementation in horticulture and landscaping, especially the genotypes with a pyramidal tree shape (E g g e n s et al., 1972;Kov a c e v i c et al., 2010b).Since the propagation of this species by stem cuttings is rather difficult and is one of the main obstacles for wider growing of this species, there is a considerable interest in its propagation by tissue culture (A h u j a , 1984; G u z i n a et al., 1986).In addition, white poplar (Populus alba L.) is one of the most interesting model tree species in biotechnology, where the tissue culture is an important propagation technique (C o n f a l o n i e r i et al., 2000).It is known that different genotypes of the same species differ in their requirements for growth in in vitro conditions.Thus, it is necessary to search for optimal culture conditions, in order to achieve satisfactory micropropagation rates and improve and optimize white poplar tissue culture procedures.
Medium pH is one of the important factors of physico-chemical environment during the development of plant tissues in vitro.It influences the utilization of medium components such as macro-and microelements and growth regulators (D e K l e r k et al., 2008).The optimum pH of in vitro medium for different phases of the morphogenesis of woody species varies (S a b o r i o et al., 1997).However, the most effective pH value is specific to the individual plant species and even cultivars, and has to be determined experimentally (O s t r o l u c k á et al., 2010; R u z i c , C e r o v i c , 2001).Tissue culture has also been extensively used to evaluate the abiotic stress tolerance of many species, since responses are relatively fast, the generation times are short, and the environment is controlled (C u i et al., 2010, L o k h a n d e et al., 2010).It is a good tool to test the in vitro ability of the species to form roots and growth in the low pH conditions as most of the factors affecting in vitro growth are similar to those limiting the growth in vivo (Kov a c e v i c et al., 2010a).Thus, the effect of low medium pH on in vitro growth will be useful to understand their tolerance to low pH, without the interference of indirect factors.
In this research, the growth and development of shoots and roots of five different white poplar genotypes were observed with the aim to optimize the culture conditions for plant propagation.In addition, the ability of the tested genotypes to grow in vitro on media with low initial pH was investigated.

Plant material
Five white poplar genotypes, considered interesting for wood production, horticulture and landscaping, were used in the experiment (Tab.1).Shoots of all five tested genotypes were multiplied by culture of axillary buds, as described by Ahuja (1984).The cultures were sub-cultured at 4-week intervals and kept at 26±2 ºC, under a 16 h photoperiod (cool white fluorescent lamps, 3500 lx), till their use in the experiment.

Media preparation
At the initial stages of the experiment, the media were sterilized by autoclaving at 120 o C and 1.1 bar for 25 min.As there were problems with the solidification of the media with low initial pH (pH 3.0 and 4.0), the sterilization of growing media in a microwave oven was tested.250 ml of the growing medium was poured into 400 ml Erlenmeyer flasks, sterilized at 800 W for 5 min, and then poured into autoclaved empty 190 ml jars.Agar and Gelrite in different concentrations were used as gelling agents (Tab.2).As there were no problems with the solidification of microwaved low pH media, all media used in the experiment were sterilized in a microwave oven.Mineral growing medium ACM (Aspen Culture Medium) described by Ahuja (1984), supplemented with 9gL -1 agar, 20gL -1 sucrose and with no growing hormones in it, was used in the experiment.The pH of the medium was adjusted to 3.0, 4.0 or 5.5 before sterilization.

Influence of medium pH on shoot and root growth
In order to investigate the effect of initial medium pH on the shoot and root growth of the tested genotypes, 1.0-1.5 cm long shoot tips of the previously multiplied shoots were placed on the media with different initial pH values: pH 3.0, pH 4.0 and pH 5.5 (control).Five shoot tips were cultured per 190 ml jar with 25 mL of culture medium.There were three jars per examined medium within a genotype.The cultures were kept at 26±2 ºC in the white fluorescent light (3500 lx) with a 16-hour photoperiod.
After 35 days of culture, the shoot and root growth was assessed for shoot height (SH), the number of roots per rooted shoot (RN), the length of the longest root (LLR), the fresh shoot mass per jar (FM) and dry shoot mass per jar (DM).

Changes in media pH
For the purpose of determining the changes in media pH, the pH of the media was measured for each jar separately after 35 days of culture (final pH).The medium in the jars was squashed manually with a plastic fork and then mixed with a similar quantity of deionised water on a magnetic stirrer for 3 min.After that, the pH was measured by inserting a pH electrode in the obtained emulsion.

Statistical analysis
The examined characters were measured either per jar (FM, DM and final pH) or the average value per jar was calculated after the measurement of individual plants (SH, RN and LLR).There were three repetitions within each genotype for each tested initial pH.The design of the experiment was totally randomized.The number of roots was transformed by square transformation ( x+1) in order to meet the normal distribution of frequencies.The obtained data were analysed by the two-way ANOVA and an LSD test with the STATISTICA 10 (StatSoft Inc., 2011) statistical program.

Media preparation
There were problems with the solidification of the autoclaved low pH media, especially when agar was used as a gelling agent.The autoclaved pH 3.0 medium with agar could not solidify, and the pH 4.0 medium was not sufficiently solid (Tab.2).Better results were obtained with the Gelrite, but with the concentrations that were several times higher than the recommended one.On the other hand, microwaved media solidified well, regardless of the pH or the gelling agent (Tab.2).That is why autoclaving was completely replaced by microwave oven sterilization, while agar was used for the solidification of media for shoot and root growth assessment.The number of roots varied among genotypes from 3.31 (KA1) to 5.59 (L-12) roots per shoot, the length of the longest root from 26.64 mm (KA1) to 41.72 mm (L-80) and shoot height from 36.21 mm (L-80) to 71.47 mm (KA1) (Tab.3).The LSD-test showed that there were no significant differences between the pH 4.0 medium and the control for all examined morphological traits in all genotypes (Tab.4).The medium with the initial pH 3.0 did not have significant effect on the number of roots and shoot height in all tested genotypes, while the positive effect of the same medium was observed for length of the longest root in Villafranca and L80, fresh mass in LBM, Villafranca and KA1, as well as for dry mass in KA1.The only negative effect of the initial pH 3.0 was recorded on the length of the longest root in KA1.Overall, the initial pH 3.0 medium had a mostly significant positive effect on all the examined traits except for the number of roots.pH.However, in our work, the genotype KA1 showed the opposite reaction, suggesting significant variability within the species.Generally, shoot height was significantly higher on the 3.0 pH medium, while there were no significant differences between the 4.0 pH and the control (pH 5.5).Martins et al. (2011) did not observe any significant differences in the shoot length of P. algarbiensis and P. almogravensis grown on low and normal pH media.Naik et al. (2010) observed that shoot regeneration of Bacopa monnieri was significantly affected by medium pH, obtaining the best results with pH 4.5.Culture on the pH 3.0 medium also had a stimulating effect on the fresh and dry mass produced per jar, as both fresh and dry biomass accumulation was by almost 60% higher on the medium with initial pH 3.0 than on the other two examined media.Several reasons for the stimulating effect of the low pH 3.0 on shoot and root development were suggested: • the increased initial uptake of microelements at low pH (Va n W i n k l e , P u l l m a n ( 2003 as the higher pH is restored for further optimal uptake of macronutrients.The final pH in all tested media was higher than the initial one, varying from pH 5.2 to nearly pH 6.0.The only exception was observed in the genotype KA1 on the medium with the initial pH 5.5, where pH decreased to final 5.2.This is not in agreement with the results of Wo o d w a r d et al. (2006), who observed a considerable decrement in rooting media pH in Eucalyptus marginata in vitro culture -from the initial pH 5.5 to the final pH 3.0, after 28 days of culture.M a r t i n s et al. (2011) have also found that the pH significantly decreased on the media inoculated with Plantago algarbiensis and Plantago almogravensis cultures, after 6 weeks of culture, with the exception of P.almogravensis during proliferation.
There were significant differences among the tested genotypes regarding the final pH value, and all genotypes could be divided into three groups according to the final pH.The first group comprises L-12 and LBM with final pH higher than pH 5.5, the second L-80 with final pH near 5.5, and the third Villafranca and KA1 with the final pH lower than 5.5.A shift of a a wide range of initial pH to the same final pH value was observed in Cucumis melo callus liquid culture by Skrivin et al. (1986).This was also observed in the species Plantago on a solid medium by M a r t i n s et al. (2011), who also recorded differences among the examined Plantago species in the final pH on both rooting and proliferation media.
In our work all tested white poplar genotypes were able to achieve and maintain a particular pH (from pH 5.2 to 6.0), regardless of the initial pH (from pH 3.0 to 5.5).It is well known that standard tissue culture media are poorly buffered (D e K l e r k et al., 2008;Wo o d w a r d et al., 2006;O w e n et al., 1991).Thus, it was found that medium pH could be influenced by media composition, storage conditions and autoclaving (A n d e r s o n , Ie v i n s h 2008; O we n et al., 1991; S a r m a et al., 1990).In addition, it was found that it could also be influenced by the selective uptake of anions and cations by explants (especially in the case of NH  , 1990).Furthermore, the buffering of media by plant material was well described by S h a n g et al. (1991) in cotton cell suspension culture.Our results suggest the buffering capacity of examined genotypes, but further research should be performed in order to differentiate this effect on medium pH from others.
); • the increased initial uptake of auxins at low pH, as suggested by D e K l e r k et al. (2008); • the buffering capacity of plants in vitro, as suggested by S h a n g et al. (1991), (Wo o d w a r d et al., 2006; B e n n e t t et al., 2003; S c h m i t z , L o r z S h a n g X.M., H u a n g J.Y., H a i g l e r C.H., Tr o l i n d e r N.L. (1991): Buffer capacity of cotton cells and effects of extracellular pH on growth and somatic embryogenesis in cotton cell suspensions, In Vitro Cell.Dev.-Pl.27P(3), (147-152) S k i r v i n R.M., C h u M.C., M a n n M.L., Yo u n g H., S u l l i v a n J., Fe r m a n i a n T. (1986): Stability of tissue culture medium pH as a function of autoclaving, time and cultured plant material, Plant Cell Rep. 5, (292-294) Statsoft Inc. (2011): STATISTICA (data analysis software system), version 10 Va n W i n k l e S., J o h n s o n S., P u l l m a n G.S. (2003): The impact of Gelrite and activated carbon on the elemental composition of two conifer embryogenic tissue initiation media, Plant Cell Rep. 21, (1175-1182) Va n W i n k l e S., P u l l m a n G.S. (2003):

Table 2 .
Solidification effect of gelatinous substance and means of sterilisation in low pH media

ПОЧЕТНE pH ВРЕДНОСТИ ХРАНЉИВЕ ПОДЛОГЕ НА РАСТ БЕЛЕ ТОПОЛЕ IN VITRO
The combined impact of pH and activated carbon on the elemental composition of plant tissue culture media, Plant Cell Rep. 22, (303-311) Wo o d w a r d A.J., B e n n e t t I.J., P u s s w o n g e S. (2006): The effect of nitrogen source and concentration, medium pH and buffering on in vitro shoot growth and rooting in Eucaliptus marginata, Sci.Hortc.110, (208-213) Yo u s s e f E.M.A., A m i n G.A. (2001): Microwave sterilization of tissue culture media, Acta Hortc.560, (513-516)