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Analysis of Flowering Ability of Regenerated Carlina Acaulis Subsp

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Analysis of Flowering Ability of Regenerated Carlina Acaulis Subsp ACTA AGROBOTANICA Vol. 60 (2): 39–44 2007 ANALYSIS OF FLOWERING ABILITY OF REGENERATED CARLINA ACAULIS SUBSP. SIMPLEX PLANTS. Alina Trejgell, Andrzej Tretyn Department of Biotechnology, Nicolaus Copernicus University, Gagarina 9, 87-100 Toruń, Poland [email protected] Received: 14.09.2007 Summary buds develop instead of leaves and axillary buds primor- Regenerated plants of Carlina acaulis subsp. simplex indu- dials (Bernier et al. 1993). In rosette forming plants, ced on shoot tips and fragments of hypocotyls, cotyledons and ro- the subapical part of meristem is activated, which brings ots were used as an experimental material. Explants were isolated about infl orescence stem development. Flowering is from 10-day-old, sterile seedlings and were put on growth me- a complex morphogenetic process, controlled by both dia supplemented with BA (3 mgdm-3), and NAA (0,1 mgdm-3). endogenous (genetic) and exogenous (light, temperatu- Plantlets were acclimatized to ex vitro conditions and planted to the re) factors (Bernier, 1988; Burn et al. 1993; K i - fi eld. Analysis of fl owering ability, infl orescence stem morphology, net, 1993; Hayama and Coupland, 2003; M o - and survival level was the objective of the study. uradov et al. 2002). Flowering may also be initiated The plants regenerated from shoot tips and cotyledons were able to fl ower in the fi rst year after acclimatization, however by stress conditions, such as drought, nutrient defi cit, or no vital seeds were found, while in the case of hypocotyl- and ro- plant density (Leavy and Dean, 1998). Readiness ot-regenerated plants fl owering appeared in the second year after to fl ower is connected with reaching some minimal size acclimatization. Number of fl owering-able plants grew in time, re- by a plant. The size may be displayed as an individual aching 100% level. Few percent of infl orescence stems displayed weight or number of leaves (Mitka and T umidaje- branches ending with additional capitula. The number of this type wicz, 1993; Klinkhamer et al. 1991). The genera- of plants decreased in successive years, while the average length of tive phase ends with the production of seeds. The seed, infl orescence stem increased. In the case of intensively fl owering and consequently embryo, development is linked to the plants, the survival rate decreased in 3 consecutive years. infl ow of many organic and inorganic compounds to the seed and accumulation of these compounds therein. All Key words: Carlina acaulis, seedling explants, infl orescence these processes are a considerable energetic burden, and stem morphology, survival plants in vivo require effi cient photosynthetic systems. Analysis of fl owering ability of regenerated Car- lina acaulis subsp. simplex plants was the objective of INTRODUCTION the work. The development of an effi cient regeneration In most of perennial plants, the process of repro- system and obtaining fl owering-competent and viable duction may take two forms: vegetative reproduction seeds-producing plants may be one of the key elements and generative reproduction (Eckert, 2002). Vegeta- in the protection of this species. The northern bor- tive reproduction means the production of novel indivi- der of Carlina acaulis incidence runs through Poland. duals off the maternal organism without zygote stadium. C. acaulis is a photophilous plant, tolerating occasio- In this case, specimens obtained (clones) are genetically nal shade, and preferring dry soils. The fl owering period identical to the maternal organism, and, as a rule, they occurs in late July-early August. In the past, this species develop and fl ower more quickly than the ones ob- was frequently harvested for use in traditional medicine tained from seeds. The plants created in this way may and as a decoration. This plant contains tannins, inuline, stay connected with the maternal plant or may separa- etheric oils and resin, which made it useful in medicine. te (Lewak, 2002). Generative reproduction increases Harvesting decreased the population size, and in certain genetic diversity in a given population. The generative locations the plant became totally extinct. Economic phase begins with a “ready to fl ower” state, which leads activity (such as marble mining) and plant succession on to changes of differentiation pattern and activity of api- meadows are further dangers for C. acaulis (P i ę koś - cal meristem. Within the shoot apical meristem, fl ower Mirkowa and Mirek, 2003). 40 Alina Trejgell, Andrzej Tretyn MATERIALS AND METHODS 74 mm for plants regenerated from shoot tips and co- tyledons, respectively. 53% of plants fell to the group Regenerated plants of C. acaulis subsp. simplex with the stem length of 0-30 mm (Fig. 2). There were were experimental material. The seeds (they come from no viable seeds in harvested achenes, which could have the Botanical Garden of Lviv National Ivan Franko Uni- been caused by the delayed fl owering period (P i ę - versity) were surface sterilized in 70% EtOH for 30 s, koś -Mirkowa and Mirek, 2003) and, conseque- then in 20% sodium hypochlorite (Domestos) for 20 ntly, a reduced number of pollinators. Alternatively, this minutes. After sterilization the seeds were rinsed four ti- might have been due to underdevelopment of seeds. mes with sterile water and placed on MS medium (M u - The plants regenerated from fragments of hy- rashige and Skoog, 1962) supplemented with GA 3 pocotyls and roots did not fl ower in the fi rst year after (1 mg dm-3). Explants were isolated from 10-day old acclimatization. The hypocotyl-regenerants display- seedlings (shoot tips, fragments of hypocotyls, cotyle- ed a strongly developed, single, erect stem, while the dons, and basal part of roots), and exposed (30 explants root-regenerants grew much slower, produced a smaller of each type) on micropropagation medium MS supple- number of leaves, and juvenile leaves (with undivided mented with sucrose (3%), agar (0,6%) and growth re- blade) lasted for a long time. One hundred percent of gulators: BA (3 mgdm-3), and NAA (0.1 mgdm-3), pH 5,7. The explants were cultivated in continuous white plants survived after four months acclimatization. (Tab. 1). Previous studies showed a similar survival level for fl uorescent light (45 μmolm-2s-1) and at 26±1°C. After four weeks forming shoots were isolated and transfer- other species of Asteraceae family, for example about red to the fresh medium. Adventitious shoots from the 90% of plantles of Echinacea purpurea (Choffe et al. 3rd subculture were rooted on the MS medium without 2000) and Achillea fi lipendulina (Evenor and Reu- growth regulators for four weeks. The plantlets were veni, 2004) and 85% of rooted plant Anthemis nobilis planted in pots fi lled with vermiculite and sand mixture survived (Echeverrigaray et al. 2000) The plants (1:1) and acclimatized to the ex vitro conditions for 4 revealed no abnormal morphological traits (leaf and fl o- weeks in a greenhouse. In May the plants were replan- wer shape) and fl owered in the typical period. In case ted to the fi eld and their fl owering ability, infl orescence of hardening plantlets of Centaurea paui, a lower sur- stem morphology, and survival level were analyzed in vival rate (70%) was obtained, but the plants grew nor- three consecutive vegetation cycles (30-40 plants rege- mally to maturity (Cuenca et al. 1999). Nevertheless, nerated from: shoot tips, hypocotyls, cotyledons, and morphological differences are sometimes observed be- 15-20 from roots). Analysis was performed when the tween regenerated and seed started plants. It was the case youngest fl owers in capitula were open (in 1st year in for Eclipa alba where the plants coming from seeds had October, 2nd and 3rd in August (or November/December a trailing habit with small leaves, while the in vitro cul- in case of the plants regenerated from hypocotyl and tured ones were erect and had larger leaves (Dhaka root in 2nd year). The vitality of seeds was analysed on and Kothari, 2005). -3 In the second year after acclimatization, the num- the MS medium supplemented with GA3 (1 mgdm ) and in the fi eld conditions. Additionally, there were ana- ber of fl owering-competent plants among those regene- lysed morphological traits and the fl owering period of rated from shoot tips and cotyledons increased to 85% the F1 generation plants. Results were expressed as per- and 100%, respectively (Tab. 1). Flowering occurred in centage (fl owering ability) or mean values and standard August and September, which is the same as in naturally errors (stem length and diameter of capitula). 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, respective- RESULTS AND DISCUSSION ly. However, in the latter case it was delayed and occurred The plants originating from shoot tips and co- in late autumn (late October and November), additionally tyledons grew intensively, a strong growth of maternal the capitula of the root-regenerated plants were immatu- rosette and the development of 2 to 5 adventitious stems re. The diameter of capitula fl uctuated between 79 and per individual were observed. Moreover, 54% of shoot 93 mm, but differences were not signifi cant. The length tip-regenerants and 17% of cotyledons-regenerants were of the infl orescence stem varied much more than in the able to fl ower in October during the fi rst year after accli- fi rst year (5-300 mm), with the average 84 and 92 mm for matization (Tab. 1). Among those, 69% plants displayed the plantlets from shoot tips and cotyledons, respectively. the typical infl orescence stem morphology (stem with These values did not exceed those typical for the species. a single capitula at the tip) (Fig.
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