ANALYSIS OF FLOWERING ABILITY OF REGENERATED CARLINA ACAULIS SUBSP . SIMPLEX PLANTS

Regenerated plants of Carlina acaulis subsp. simplex induced on shoot tips and fragments of hypocotyls, cotyledons and roots were used as an experimental material. Explants were isolated from 10-day-old, sterile seedlings and were put on growth media supplemented with BA (3 mg dm-3), and NAA (0,1 mg dm-3). Plantlets were acclimatized to ex vitro conditions and planted to the fi eld. Analysis of fl owering ability, infl orescence stem morphology, and survival level was the objective of the study. The plants regenerated from shoot tips and cotyledons were able to fl ower in the fi rst year after acclimatization, however no vital seeds were found, while in the case of hypocotyland root-regenerated plants fl owering appeared in the second year after acclimatization. Number of fl owering-able plants grew in time, reaching 100% level. Few percent of infl orescence stems displayed branches ending with additional capitula. The number of this type of plants decreased in successive years, while the average length of infl orescence stem increased. In the case of intensively fl owering plants, the survival rate decreased in 3 consecutive years.


INTRODUCTION
In most of perennial plants, the process of reproduction may take two forms: vegetative reproduction and generative reproduction (E c k e r t , 2002).Vegetative reproduction means the production of novel individuals off the maternal organism without zygote stadium.In this case, specimens obtained (clones) are genetically identical to the maternal organism, and, as a rule, they develop and fl ower more quickly than the ones obtained from seeds.The plants created in this way may stay connected with the maternal plant or may separate (L e w a k , 2002).Generative reproduction increases genetic diversity in a given population.The generative phase begins with a "ready to fl ower" state, which leads to changes of differentiation pattern and activity of apical meristem.Within the shoot apical meristem, fl ower buds develop instead of leaves and axillary buds primordials (B e r n i e r et al. 1993).In rosette forming plants, the subapical part of meristem is activated, which brings about infl orescence stem development.Flowering is a complex morphogenetic process, controlled by both endogenous (genetic) and exogenous (light, temperature) factors (B e r n i e r , 1988; B u r n et al. 1993;K in e t , 1993;Hayama and C o u p l a n d , 2003;M ou r a d o v et al. 2002).Flowering may also be initiated by stress conditions, such as drought, nutrient defi cit, or plant density (L e a v y and D e a n , 1998).Readiness to fl ower is connected with reaching some minimal size by a plant.The size may be displayed as an individual weight or number of leaves (M i t k a and T u m i d a j ew i c z , 1993; K l i n k h a m e r et al. 1991).The generative phase ends with the production of seeds.The seed, and consequently embryo, development is linked to the infl ow of many organic and inorganic compounds to the seed and accumulation of these compounds therein.All these processes are a considerable energetic burden, and require effi cient photosynthetic systems.
Analysis of fl owering ability of regenerated Carlina acaulis subsp.simplex plants was the objective of the work.The development of an effi cient regeneration system and obtaining fl owering-competent and viable seeds-producing plants may be one of the key elements in the protection of this species.The northern border of Carlina acaulis incidence runs through Poland.C. acaulis is a photophilous plant, tolerating occasional shade, and preferring dry soils.The fl owering period occurs in late July-early August.In the past, this species was frequently harvested for use in traditional medicine and as a decoration.This plant contains tannins, inuline, etheric oils and resin, which made it useful in medicine.Harvesting decreased the population size, and in certain locations the plant became totally extinct.Economic activity (such as marble mining) and plant succession on meadows are further dangers for C. acaulis (P i ę k o ś -M i r k o w a and M i r e k , 2003).

MATERIALS AND METHODS
Regenerated plants of C. acaulis subsp.simplex were experimental material.The seeds (they come from the Botanical Garden of Lviv National Ivan Franko University) were surface sterilized in 70% EtOH for 30 s, then in 20% sodium hypochlorite (Domestos) for 20 minutes.After sterilization the seeds were rinsed four times with sterile water and placed on MS medium (M ur a s h i g e and S k o o g , 1962) supplemented with GA 3 (1 mg dm -3 ).Explants were isolated from 10-day old seedlings (shoot tips, fragments of hypocotyls, cotyledons, and basal part of roots), and exposed (30 explants of each type) on micropropagation medium MS supplemented with sucrose (3%), agar (0,6%) and growth regulators: BA (3 mg dm -3 ), and NAA (0.1 mg dm -3 ), pH 5,7.The explants were cultivated in continuous white fl uorescent light (45 μmol m -2 s -1 ) and at 26±1°C.After four weeks forming shoots were isolated and transferred to the fresh medium.Adventitious shoots from the 3 rd subculture were rooted on the MS medium without growth regulators for four weeks.The plantlets were planted in pots fi lled with vermiculite and sand mixture (1:1) and acclimatized to the ex vitro conditions for 4 weeks in a greenhouse.In May the plants were replanted to the fi eld and their fl owering ability, infl orescence stem morphology, and survival level were analyzed in three consecutive vegetation cycles (30-40 plants regenerated from: shoot tips, hypocotyls, cotyledons, and 15-20 from roots).Analysis was performed when the youngest fl owers in capitula were open (in 1 st year in October, 2 nd and 3 rd in August (or November/December in case of the plants regenerated from hypocotyl and root in 2 nd year).The vitality of seeds was analysed on the MS medium supplemented with GA 3 (1 mg dm -3 ) and in the fi eld conditions.Additionally, there were analysed morphological traits and the fl owering period of the F1 generation plants.Results were expressed as percentage (fl owering ability) or mean values and standard errors (stem length and diameter of capitula).

RESULTS AND DISCUSSION
The plants originating from shoot tips and cotyledons grew intensively, a strong growth of maternal rosette and the development of 2 to 5 adventitious stems per individual were observed.Moreover, 54% of shoot tip-regenerants and 17% of cotyledons-regenerants were able to fl ower in October during the fi rst year after acclimatization (Tab.1).Among those, 69% plants displayed the typical infl orescence stem morphology (stem with a single capitula at the tip) (Fig. 1A), while 8% bore one or two additional capitula and 23 % carried three or more (Fig. 1B).The diameter of capitula fl uctuated around 80 mm.The infl orescence stem length was in the range of 0 to 120 mm, with the mean value of 26 mm and 74 mm for plants regenerated from shoot tips and cotyledons, respectively.53% of plants fell to the group with the stem length of 0-30 mm (Fig. 2).There were no viable seeds in harvested achenes, which could have been caused by the delayed fl owering period ( P i ęk o ś -M i r k o w a and M i r e k , 2003) and, consequently, a reduced number of pollinators.Alternatively, this might have been due to underdevelopment of seeds.
The plants regenerated from fragments of hypocotyls and roots did not fl ower in the fi rst year after acclimatization.The hypocotyl-regenerants displayed a strongly developed, single, erect stem, while the root-regenerants grew much slower, produced a smaller number of leaves, and juvenile leaves (with undivided blade) lasted for a long time.One hundred percent of plants survived after four months acclimatization.(Tab.1).Previous studies showed a similar survival level for other species of Asteraceae family, for example about 90% of plantles of Echinacea purpurea (C h o f f e et al. 2000) and Achillea fi lipendulina (E v e n o r and R e uv e n i , 2004) and 85% of rooted plant Anthemis nobilis survived (E c h e v e r r i g a r a y et al. 2000) The plants revealed no abnormal morphological traits (leaf and fl ower shape) and fl owered in the typical period.In case of hardening plantlets of Centaurea paui, a lower survival rate (70%) was obtained, but the plants grew normally to maturity (C u e n c a et al. 1999).Nevertheless, morphological differences are sometimes observed between regenerated and seed started plants.It was the case for Eclipa alba where the plants coming from seeds had a trailing habit with small leaves, while the in vitro cultured ones were erect and had larger leaves (D h a k a and K o t h a r i , 2005).
In the second year after acclimatization, the number of fl owering-competent plants among those regenerated from shoot tips and cotyledons increased to 85% and 100%, respectively (Tab.1).Flowering occurred in August and September, which is the same as in naturally growing plants.Harvested seeds were viable.Moreover, the fl owering of plants regenerated from hypocotyls and roots was observed in 26%, and 20% of them, respectively.However, in the latter case it was delayed and occurred in late autumn (late October and November), additionally the capitula of the root-regenerated plants were immature.The diameter of capitula fl uctuated between 79 and 93 mm, but differences were not signifi cant.The length of the infl orescence stem varied much more than in the fi rst year (5-300 mm), with the average 84 and 92 mm for the plantlets from shoot tips and cotyledons, respectively.These values did not exceed those typical for the species.The most prominent group (27%) had the stem length between 31 and 60 mm (Fig. 1).In the case of hypocotyl-and root-regenerated plants that were fl owering for the fi rst time, the average infl orescence stem length was 50 mm and 5 mm, respectively.The number of plants Flowering ability and infl orescence stem morphology of Carlina acaulis subsp.simplex plantlets in three consecutive years after acclimatization.
bearing branched stems was signifi cantly reduced in comparison to the previous year (Tab.1).The survival level for the second year after acclimatization was 100% in the case of the plants regenerated from shoot tips and cotyledons, while for the hypocotyl-, and root-regenerated ones it was 93.8% and 87.5%, respectively (Tab.1).
In the third year all the plants were able to fl ower, regardless of their origin.The number of individuals carrying branched infl orescence stems was less than 3% (Tab.1).The diameter of capitula fl uctuated between 81 and 95 mm.We did not observe signifi cant differences during 3 years of observation.The stem length varied between 50 and 280 mm, and the group with the length over 210 mm was the biggest one (Fig. 1).In the third year of culture in soil, 93% of shoot tips plantlets, 90% of those regenerated from cotyledons and 100% of the remaining two groups survived.
The observed changes in infl orescence stem development (inhibition of growth on length and branching) in the fi rst year after in vitro culture may be a result of exogenous benzylaminopurine in regeneration media and its elevated level in plant tissues.It is known that cytokinins cancel shoot tip domination, which manifests in the shortening of stems and activation of axillary buds (K a k i m o t o , 1996).In the fourth year of soil culture, only 40% of the plants regenerated from shoot tips survived.The obtained achenes contained viable seeds, 98% of seed germinated on medium supplemented with GA 3 .At the same time, seedlings growing from seeds were observed in fi eld conditions.Those plants revealed typical morphological traits: non-branched stems and a normal fl owering period.The survival level decline is probably due to very intensive fl owering in three consecutive vegetation seasons and the dying of maternal plants that do not produce adventitious stems in the last season.One may suppose that intensive fl owering and seed production is a big energetic burden leading to senescence and dying.A similar phenomenon was reported by C z a r n e c k a (2006) in a 15-year study of the fl owering pattern of Senecio macrophyllus in nature.The absence of rosette growth was observed after a period of intensive fl owering.It shows that the individuals under study entered a static phase or started dying.

CONCLUSIONS
All plants, regardless of explant source, were fl owering-competent.
Plantlets coming from shoot tips and cotyledons were able to fl ower in the fi rst year of soil culture.
Benzylaminopurine present in the regeneration medium could have been a factor causing inhibition of length growth and branching of infl orescence stems during the two initial vegetation seasons.
Intensive fl owering might have been a cause of survival rate decrease.
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