MORPHOGENESIS OF MISCANTHUS × GIGANTEUS IN VITRO

One of the top priority tasks of all the civilized countries is ensuring the development of bioenergetics, preserving natural resources and searching for new sources of alternative energy. Recent years have witnessed a considerable interest to the new crop, Miscanthus × × giganteus, which is among the most promising plants to obtain biofuel of organic origin – ecologically pure and renewable source of energy [1]. By-products of plant origin (straw, corn stalks, etc.) and dedicated energy plants may also be used as energy commodity [2, 3]. One of these plants is a tree-like grass – miscanthus. Miscanthus × giganteus, an allotriploid hybrid, remarkable for a considerably higher biomass gain compared to other species, is of the highest interest for biofuel production. This crop has a number of advantages compared to other energy crops: high biomass performance, positive energy balance, easy maintenance in soil, drought resistance [4].


INTRODUCTION
One of the top priority tasks of all the civilized countries is ensuring the development of bioenergetics, preserving natural resources and searching for new sources of alternative energy.Recent years have witnessed a considerable interest to the new crop, Miscanthus × × giganteus, which is among the most promising plants to obtain biofuel of organic origin -ecologically pure and renewable source of energy [1].By-products of plant origin (straw, corn stalks, etc.) and dedicated energy plants may also be used as energy commodity [2,3].One of these plants is a tree-like grass -miscanthus.Miscanthus × giganteus, an allotriploid hybrid, remarkable for a considerably higher biomass gain compared to other species, is of the highest interest for biofuel production.This crop has a number of advantages compared to other energy crops: high biomass performance, positive energy balance, easy maintenance in soil, drought resistance [4].
Miscanthus × giganteus is a large warm-season Asian grass and a novel leading bioenergy crop in Asia, Europe and North America [5,6].The experience of Europe demonstrates that miscanthus has high biological performance of biomass in a wide geographical range of moderate climatic regions, including marginal lands [7,8].It is not grown much in Ukraine yet, however, the interest to this energy crop is increasing consistently, as it can yield up to 20 t/ha of dry matter in favorable weather conditions. of current species from the standpoint of using them as bioenergetics commodity [9][10][11].In order to accelerate the selection process, there are biotechnology methods, used to obtain a suf¿ cient amount of material for studies in a short period of time [12].Solving this problem is related to the search for the ways of direct morphogenesis in vitro in the culture medium and the possibility of regulating these ways in the controlled experimental conditions in vitro.

The aim of the work.
To obtain planting material of Miscanthus × giganteus in a large amount in order to study the peculiarities of morphogenesis of isolated meristem culture.

MATERIALS AND METHODS
The material of the study was dormant buds of 3-yearold rhizomes of Miscanthus × giganteus.70 % ethanol and 0.1 % solution of HgCl 2 were used for sterilization.The explants were sterilized for 1.5 min in 70 % ethanol and for 22 min in the solution of 0.1 % mercury bichloride with subsequent washing in three portions of water for 7-10 min.Sterile explants were placed into tubes with 10 ml Murashige and Skoog (MS) nonhormone culture medium [13] with subsequent transfer to the modi¿ ed culture MS medium: MSR1 (MS + + BAP (0.2 mg/l)) and MSR2 (MS + BAP (0.75 mg/l) + + kinetin (1.2 mg/l)).The biological replication was 40 plants.

RESULTS AND DISCUSSION
Our studies demonstrated that the highest percentage of sterile dormant buds (70 %) was obtained by keeping them consistently in 70 % C 2 H 5 OH (1.5 min) with further transfer into 0.1 % solution of HgCl 2 (22 min) and washing three times in sterile distilled water.The extracted dormant buds started enlarging on the 5 th -6 th day after planting into the culture medium.On days 8-10, they were transferred into the culture media MSR1 and MSR2.The ef¿ ciency of selected media was estimated by the following indices: the length of sprouts, their number and frequency of multiple tillering.The measurements of morphometric indices and the calculations of quantitative data were performed for 30 days (Table 1).
During the ¿ rst three weeks of cultivation, the regeneration processes in isolated miscanthus plants were most active in the presence of a larger amount of BAP  and kinetin in the culture medium.According to our observations, kinetin (1.2 mg/l) promoted the formation of buds and additional sprouts.
There is a known scienti¿ c fact that some cytokins are capable of removing apical domination and leading to awakening of accessory buds.In our studies the addition of BAP (0.75 mg/l) caused active formation of 3-7 additional sprouts on the 4 th -5 th week of cultivation.It should be noted that saccharose (30 mg/l) was found to be a favorable source of carbohydrates at this stage of cultivating isolated explants.
The studies demonstrated that the optimal culture medium for regeneration of microsprouts was MSR2, supplemented with BAP (0.75 mg/l) and kinetin (1.2 mg/l).Here the frequency of regeneration of sprouts was 90.0-100.0% with the development of the main sprout and multiple tillering with the frequency of 85.0-100.0% (Fig. 1).
Noteworthy is the rhizogenesis process.The rooting of Miscanthus × giganteus sprouts in vitro depended on the size of a sprout and the number of passages performed.The 5-6-cm-long sprouts (8-9-week-old) were transferred to MS culture medium with half-dose of macro-and microelements without growth regulators.Miscanthus × giganteus plants demonstrated their capability of normal development in this culture medium.The formation of the root system was observed on the 8-11 th day of cultivation (Fig. 2).
After the formation of the root system, the laboratory plants started growing actively, doubling and even tripling in their sizes within 7 days with active formation of leaves.There were 4-10 roots of 1.5 cm on average obtained per one sprout.On the 30 th day of cultivation the number of roots was in the range from 9 to 22, and their length was from 4 to 13 cm (Table 2).The frequency of rooting was 95.0-97.0%.The 12-14-week-old laboratory plants (depending on the cultivation period in the rooting medium) can be further grown in a greenhouse.Regenerate plants of 5-7 cm with a well-developed root system were carefully extracted with forceps, their roots were thoroughly washed from the culture medium (to prevent rotting and perishing of plants) and they were planted into different soil substrates.Three different sterile substrates were used to adapt regenerate plants: No. 1 -peat:sand:perlite (2:1:1); No. 2 -peat:sand (2:1); No. 3 -peat:perlite:soil (2:2:1) (Fig. 3).The plants were watered with distilled water regularly.
It is evident that substrate No. 1 was found to be the most ef¿ cient for miscanthus plants, as, contrary to substrates Nos. 2 and 3, it promoted fast rooting, growth and development of sprouts.Four-¿ ve weeks later the plants showed the formation of 3-4 leaves and ¿ broid root system.In these conditions the survivability of microplants was 91-95 %.Therefore, a series of studies was conducted to investigate the peculiarities of morphogenesis of Miscanthus × giganteus isolated meristems.

CONCLUSIONS
The peculiarities of morphogenesis in Miscanthus × giganteus were studied in culture in vitro.The frequency of regeneration of sprouts was 90.0-100.0%, there was noted development of the main sprout and multiple tillering with the frequency of 85.0-100.0%.The most suitable substrate for adaptation of miscanthus to in vivo conditions was peat : sand : perlite in the ratio of 2:1:1, the survivability of regenerate plants was 91-95 %.

Table 1 .
The impact of the culture medium composition on the development of Miscanthus × giganteus meristem cultures