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本頁は Jpn. J. Crop Sci. 66 : 137〜138 (1997) に掲載したものです.

Induction of Haploid Plantlets by Anther Culture of Bupleurum falcatum L.*

    Tae−Kwon SHON and Tomohiko YOSHIDA
  (Faculty of Agriculture , Kyushu University, Fukuoka 812-81, Japan)

Received June 1O,1996
ミシマサイコの葯培養による半数体植物の作出:孫 太権・吉田智彦(九州大学農学部)
キーワード:植物体再分化,ミシマサイコ,葯培養.
Keywords:Anther culture,Bupleurum falcatum L..,Plant regeneration.
* An outline of this report was presented at the 201st meeting of the Crop Science Society of Japan in April, 1996.

Bupleurum falcaium L. (Mishimasaiko in Japanese) is one of the components of Chinese crude drugs, and is widely used in painkillers, anti-allergy, anti-inflammatory and anti-pyertic medications. Breeding of B. falcalum L. for higher yield and quality is scarce. B. falcalum L. is a cross pollinating species and genetically heterozygous. Through anther culture, haploid plants can be obtained. Homozygous plants, which can be obtained by doubling their chromosome number, can be used as inbred lines in hybrid breeding. Doubled haploid plants may also contribute to the genetic studies of B. falcatum L..

Since the first paper on pollen embryogenesis in anther culture of Dature innoxia was reported by Guha et a1.4), many studies have been carried out using various plant species.

B. falcalum L. is an Umbelliferae species. For the Umbelliferae, some efforts have been made to produce haploid plants using anther in carrot (Daucus carota L.)6). There are some efforts regarding plant regeneration and callus formation through leaf and root suspention culture in B. falcatum L.1,5). There is no study of anther culture in B. falcalum L. reported, however.

This study was initiated to produce haploid plants by way of anther culture for B. falcatum L. plants originated from three different habitats.

Materials and Methods
B. falcalum L. populations which originated from Fukuoka and Kumamoto, Japan and from Jeongseon, Korea were grown at Kyushu University in a field. Anthers were obtained from July to October, 1995. Anthers from one plant were cuture on the same day. Anthers at the uninucleate stage were collected in the morning, and were surface-sterilized in ethanol, and 2% sodium hypochlorite and rinsed three times with sterile distilled water. Following the method used in carrot6), MS medium7) containing 3% sucrose and 1 mg of 2,4-D per liter adjusted to pH 5.8 was used.

The anthers were placed in a petri dish containing 20 ml of the medium and then incubated at 25℃ in the dark. After 5 weeks, one callus block of 3 mm in diameter was plated in a petri dish with 10 ml regeneration medium containing 3% sucrose without phytohormones, and cultured to induce plant regeneration at 25℃ under a 16 hour-photoperiod of 400 μmol m-2s-1. For chromosome observation, root tips were pretreated with solution of 0.1% colchicine for 2 hours at 20℃. After fixation in ethanol/acetic acid (3:1v/v) for 30 min. at 5℃, they were treated 1N HCl for 5 min. and stained in 1% aceto-orcein solution. Chromosomes of plants grown from the seed as well as anther-derived plantlets were observed.

Results and Discussion
The formation of callus was observed from 3 weeks after inoculation. A total of 4736 anthers were cultured, and a total of 990 calli were obtained. The frequency of callus formation of different geographical origins, from July 15 to August 17, is shown in Table 1. The value of callus formation based on the average frequency was 22.6% for Fukuoka, 10.0% for Kumamoto and 23.1% for Jeongseon. The values in Table 1 also show the variation among plants. The variation among plants on callus formation was high and in the case of Fukuoka, it was from 2% to 51%. Especially, the formation of calli was high for plants of July 29. It shows that differences in callus formation were observed among individual plants as well as origins.
Table 1. Callus formation of B. falcalum L. from three
     different habitats from July 15 to August 17.
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− 
  No.of calli obtained / No.of anthers cultured
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− 
 Date   Fukuoka  Kumamoto  Jeongseon
 Jul.15   2/111
    19  108/296
    22   20/333
    25   12/370
    27   51/222
    28   7/148
    29  189/370
    31   80/l85
 Aug.4   32/185
 Aug.1           14/222
     3           30/259
     9           26/222
    10                   94/518
    11                   163/481
    12                   76/444
    17                   86/370
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Mean(%)   22.6    10.0     23.1
        (2-5l)*  (6-12)*  (17-34)*
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
 *The range of frequency of callus formation.
Variations among genotypes for callus formation and plant regeneration were widely observed in many species3). In B. falcalum L. variations among genotypes (among habitats and among individual plants) were also observed. From 2 weeks after transfer to the regeneration medium, roots and leaves were induced (Fig.1) and 27 plantlets were obtained in total. These plantlets are now growing in our laboratory.

The chromosome number of B. falcatum L. was studied and three basic cytogenetic types, 2n=20, 26 and 32 have been established1,2,8) though aneuploid variations were observed. The chromosome number of many plants in our experiment was 2n=26. Fig. 2 shows that the number of chromosomes of an anther-derived plantlet was n=13, showing that this plantlet was a haploid which might be derived from a plant having 2n=26 chromosomes. We also observed aneuploid variations for anther-derived plantlets, which might be induced during callus formation as is the case observed by Amano et al.1) in the plants regenerated from callus from leaves.

This is the first report on the production of a haploid plantlet through the anther culture of B. falcatum L.. Haploid-production may offer a tool for more efficient breeding programs and genetic studies in B.falcatum L.

Fig.1. Plantlets regenerated from callus on regeneration medium.







Fig.2. Chromosomes (n=13) in a root tip cell of a regenerated plantlet.
          
        References
 1. Amano, A. et al. 1989. Shoyakugaku Zasshi 43:l3-l8.*
 2. Chung, S.H. et al. 1995. Korean J. Medicinal Crop Sci. 3 : 61-65.
 3. Collins, G.B. l977. Crop Sci. 17 : 583-586.
 4. Guha, S. et al. 1964. Nature 204:497.
 5. Hiraoka, N. et a1. 1983. Shoyakugaku Zasshi 37 :62-67.*
 6. Hu, K.L. et al. 1993. J. Jpn. Soc. Hort. Sci. 62 :561-565.
 7. Murashige, T. et al. 1962. Physiol. Plant. 15 : 473-479.
 8. Ohta, S. 1991. J. Sci. Hiroshima Univ., Ser. B, Div.2. 23 :273-348.
    * In Japanese with English Summary.

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