micropropggation of scabiosa caucasica bieb.cv.caucasica blue

MICROPROPAGATION OF SCABIOSA CAUCASICA BIEB.CV.CAUCASICA BLUE

TAKASHI HOSOKI*

AND

SACHIE NOJIMA

Faculty of Life and Environmental Science,Shimane University,1060,Nishikawatsu-cho,Matsue,Shimane Pref.690-8504,Japan

(Received 23October 2003;accepted 13May 2004;editor A.Altman)

Summary

Micropropagation of Scabiosa caucasica cv.Caucasica Blue was achieved by culturing,separating axillary and adventitious shoots,or node sectioning on Murashige and Skoog (MS)medium supplemented with benzyladenine (BA).The highest frequency of adventitious shoots regenerated from nodal or internodal explants and leaf blade (with or without petiole)appeared to occur on MS medium with 4.4and 18m M BA,respectively.Addition of 0.19or 1.9m M a -naphthaleneacetic acid to the BA-containing medium promoted callus formation and reduced shoot organogenesis.During micropropagation,shoot nodal explants derived from in vitro shoots cultured on MS medium supplemented with 4.4m M BA yielded 8.9shoots per explant within 40d after culture initiation.Key words:micropropagation;Scabiosa caucasica ;shoot regeneration;tissue culture.

Introduction

Scabiosa caucasica Bieb.is a native Caucasian annual or perennial plant with blue flowers that can grow up to 60cm tall (Bailey,1976).This plant is used for cut flowers and in ornamental gardens.One cultivar,‘Caucasica Blue’,has been cultivated as a perennial Scabiosa plant (Takii Seed Co.,Kyoto)that produces attractive large flowers.In recent years,the demand for herbaceous perennial plants has been increasing rapidly (Hayashi et al.,2001).To produce a larger number of nursery plants,conventional propagation of S.caucasica has been employed by separation of axillary shoot clusters into individual shoots.However,this method is too slow to meet the demand.

Development of an efficient tissue culture system,in which shoots or plants can be regenerated from cultured explants at high frequencies,is important for plant propagation,genetic transform-ation,and cell manipulation (Matsumoto and Yamaguchi,1991).In S.columbaria ,Romeijn and Lammeren (1999)reported shoot regeneration though callus from gametophytic cells in anthers and ovules.However,shoot regeneration from S.caucasica in vitro has not been reported.In this study,we report direct and indirect shoot regeneration from different explants such as node,internode,leaf,petiole,and petiole with leaf organs of S.caucasica cv.Caucasica Blue in vitro .A similar method has also been reported previously in Polymnia sonchifolia (yacon)(Hamada et al.,1990)and Salvia leucantha (Hosoki and Tahara,1993).

Materials and Methods

Micropropagation from shoot apices .The donor plants of S.caucasica cv.Caucasica Blue used for this study were grown under greenhouse conditions with day and night temperatures of 25and 158C,respectively.When the plants reached the 7–10-leaf stage,shoot apices (1cm long)were excised and washed in tap water containing 0.1%(v/v)Tween 20.Explants were surface-sterilized for 10min in a sodium hypochlorite solution (0.6%active chlorine),followed by two rinses in sterile water.After cutting off a few basal leaves,shoot tips (about 0.5cm long)were excised and transferred to test tubes (15£2cm),each containing 15ml of solidified medium,and sealed with aluminum foil.The culture medium consisted of MS (Murashige and Skoog,1962)major salts,Ringe and Nitsch (1968)minor salts and vitamins,2%(w/v)sucrose and 0.8%agar supplemented with 0,0.44,or 4.4m M benzyladenine (BA).The pH was adjusted to 5.6with NaOH and the medium was autoclaved for 15min at 1208C.Shoots were multiplied with five subcultures at 3-wk intervals by separation of axillary or adventitious shoots or by node cutting when internodes were elongated.Cultures were kept at 25^18C under a 16-h photoperiod with a light intensity of 52m mol m 22s 21provided with fluorescent lamps.

For root induction,shoots (about 3cm long)were transferred to 0.2%(w/v)Hyponex (6.5N–6P–19K)(Hyponex Corp.,Ohio)medium containing vitamins and minor salts (Ringe and Nitsch,1958)and basal MS medium in the presence of 4.9m M indole-3-butyric acid (IBA).Rooting response in terms of rooting frequency,number and length of roots per explant was evaluated after 4wk in culture.

For acclimatization,20rooted shoots were transferred to pots (6cm diameter)containing sandy loam and part bark mix (2:1,v/v)and covered with plastic bags to maintain high humidity.The bag was gradually opened daily for 20d to reduce the humidity.The temperature was also lowered to 208C during acclimatization to inhibit fungal and bacterial growth.

Adventitious shoot formation .To investigate the capacity of shoot formation,four types of explant were prepared from shoots grown for 1mo.on MS medium containing 0.44m M BA.These explants included:(1)1cm blades that were derived from leaves after both proximal and distal ends were removed;(2)intact blades with petioles,with petioles inserted about 0.8cm deep into the medium;(3)0.5cm long nodal segments,with segments inserted 2mm deep into the medium;and (4)0.5cm long internodal segments.These explants were cultured on basal MS medium supplemented with 0,0.19,or 1.9m M a -naphthaleneacetic acid (NAA)in combination with 4.4,18,or 35m M BA.Each treatment consisted of 10explants and experiments were repeated once.Data were pooled and analyzed statistically by ANOVA and Tukey’s test.

Results and Discussion

Micropropagation from shoot apices .‘Caucasica Blue’grown in vitro showed poor stem elongation (data not shown)with leaves *Author to whom correspondence should be addressed:Email hosoki@life.shimane-u.ac.jp

In Vitro Cell.Dev.Biol.—Plant 40:482–484,September–October 2004DOI:10.1079/IVP2004572

q 2004Society for In Vitro Biology 1054-5476/04$18.00+0.00

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