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Plant Cell Reports (1994) 14:65-68

~, Springer-Verlag1994

Plant Cell Reports

High rate of virus-free plantlet regeneration via garlic scape-tip culture


Yun Ma 1, Hong-Long Wang 1, ,, Cun-Jin Zhang

and Yu-Qing Kang 2

Department of Biology, Tianjin Normal University, Tianjin 300074, China 2 Center of Laboratories, Tianjin Institute of Education, Tianjin 300020, China * Present address." INRA, Symbiotes des Racines, 34060 Montpellier, France Received 11 September 1993/Revised version received 28 March 1994 Communicated by A. Komamine

ABSTRACT
Shoot cultures of Allium sativum L. were established from stem-tip and scape-tip. Various combinations of media, growth regulators were compared. The best establishment and proliferation of stem-tip and scape-tip explants were obtained on MS medium containing 0.5 mg Ia NAA and 0.5 mg 1-t KT. The roots were produced and the bulblets were formed after the micro-shoots were transferred to the hormone-free MS medium. The virus of each tube-plant was detected by ELISA. The virus elimination rates of plantlets from stem-tip with 3 leaf primordia, with 1-2 leaf primordia, and scape-tip were 26.5%, 45.4%, and 77.6% respectively. The procedure of scape-tip culture would be applied for virus elimination to the species propagated vegetatively. Key words: Garlic, Scape tip culture, Stem tip culture, Virus elimination, ELISA INTRODUCTION Garlic (Allium sativum L.) is one of the most widely used species in food processing, chinese medicine, culinary preparations and as a condiment. Most commercial garlic varieties are virus infected, Further, the plant is sexually sterile and propagated strictly by cloves and bulbs. As a result, virus infections are always transferred by this vegetative propagation. The Garlic Mosaic Virus (GMV) causes a poor quality product and reduces the plant production(Ma et al. 1991). Clearly, shoot tip culture provides a means for virus-free plant production and mass ctonal propagation.
Correspondence to: H.L. Wang

Plantlet regeneration from callus culture has been reported(He et al. 1990; Wang et al. 1991a, 1992; Nagakubo et al. 1993). Somatic embryogenesis has been sucessful(Abo EI-Nil 1977; Wang et al. 1994). There are some reports on virus-free plantlet production through shoot-tip culture of garlic(Abo El-Nil 1977; Sant 1980), but the virus elimination rate is lower and the methods of virus detection are inaccurate and time consuming. This report deals with the high rate Virus-free plantlet production via garlic scape-tip culture and an ELISA method tor virus detection. M A T E R I A L S AND M E T H O D S Tissue culture Scapes were collected during the period of garlic reoroductive ~,-rowth. Scaoes and sprouting Bodi-~own garlic cloves variety "Red Six Cloves" were surface sterilized in 1.0% sodium hypochlorite solution for 15 rain and washed 3 times with sterile waten Stem tips without a leaf primordium(<0.3mm), with 1-2 leaf primordia(0.3-0.5mm), and with t h r e e l e a f primordia(>0.5mm)., and scape tips(2-3mm) were excised aseptically with the aid of a binocular microscope and cultured on the different media. Two different basal media namely MS and B5 supplemented with GA, IAA and NAA(0.1-1.0 mg t1) and KT, 6-BA and 2,4-D(0.522.0 mg 1-1) in combinations were used. The media were adjusted to pHS.8, 0.8% agar and 0.3% sucrose were added before sterilization. The composition of the different media used are as follows(concentrations in mg !-~):

66 M1 .... MS + 2,4-D(2.0) + KT(0.5) M2 .... MS + NAA(0.5) + KT(0.5) M3 .... MS + NAA(1.0) + KT(1.0) M4 .... MS + NAA(0.1) + KT(1.0) M5 .... MS + IAA(I.0) + KT(1.0) M6---B5 + NAA(0.1) + 6-BA(1.0) M7---B5 + NAA(0.1) + KT(I.0) M8 .... MS + NAA(0.3) + GA(0.3) Each tip was cultured in a glass tube (20 mm X 200ram) containing 20 ml of sterilized solid media. The cultures were incubated at a temperature of 25+1~ under 16 h daily illumination with white fluorescent light(I,500 Ix) (He et al. 1990; Wang et al. 1992, 1994).

RESULTS AND DISCUSSION


In preliminary experiments, 0.3-0.5 mm shoot tips from garlic cloves were used. Among the two different basal media used, MS and B5 were found to be better for multiple shoot induction, respectively. For shoot proliferation, the use of single auxin or cytokinin was not effective, hence combinations of auxins with cytokinins were used. The morphogenetic responses of shoot tips cultured on MS and B5 media supplemented with auxin and cytokinin combinations are summerized in Table 1. Shoot growth and proliferation, which started as leaf primordium elongation, were followed by shoot sprouting and light green protuberances appearing from the base of the explants. Shoots 3-6 cm in length and 2-7 buds per shoot were observed after 50 days incubation. NAA was found to be most effctive, the KT, 6-BA , seemed to have stimulatory effect on shoot proliferation. On M8 medium, only a single elongated shoot of 7-8 cm in length was oberserved in 50-60 days. So cytokinins were necessary for the speedy shoot proliferation. 2,4-D exhibited a strong dedifferentiation effect. These results are in agreement with other reports on garlic shoot tip culture( Wang & Huang 1974; Sant 1980). High concentration of NAA(1.0 mg 1-1)resulted in reducing the growth of shoots and making the leaf primordium elongating and curling. Low auxin with high concentratioins of cytokinins(a ratio of 1:10) resulted in shoot ~owth and proliferation. But the shoots were thick and short, at the same time, the shoot multiplication rate was lower. It was also noted that the auxins and cytokinins with low concentrations(NAA 0.5 mg 1 + -I KT 0.5 mg I-I) improved the quality and quantity of the shoots. After identifying the optimum growth regulator levels and the basal media for the induction of multiple shoots, different explants such as stem tips without a leaf primordium, with 1-2 leaf primordia, with 3 leaf primordia and scape tips were studied to improve the rate of virus-free plantlet production. Stem tips(<0.3 ram, 0.3-0.5 ram, and >0.5 mm) and scape tips(Fig. A) were cultured on M2 medium. Stem tips(<0.3 ram) were not shoot sprouting and formed calli after 30-40 days incubation. It was suggested that shoot growth was impossible from insufficient size of shoottip. Stem tips(0.3-0.5 ram, >0.5 ram) developed 6+1 micro-shoots per shoot within 50-day on M2 medium. Scape tips producexi 5 micro-shoots per shoot on M2

Virus purification
The garlic mosaic virus (GMV) was partially purified from garlic leaves showing typical mosaic symptoms according to the methods described by Mohamed & Young (1981) and Ma et al. (1991).

Antiserum production
Antiserum was produced in a rabbit by two subcutaneous injections followed by two intramuscular injections at 7-d intervals with approx. 1 mg purified virus mixed with an equal volume of Freund's incomplete adjuvant. Antiserum was collected 10 days after the third injection. The titre was 1250.

Extraction of samples
Leaf samples were extracted by grinding 50 mg of leaf from the cultured plantlets in a pestle with 250ml 0.1 M phosphate buffer pH 7.4 containing 10% Tween 20 (PBST). Further dilutions were made in PBST.

ELISA
Tests were done essentially as described by Clark & Adames (1977) except that the incubations with immunoglobulin and enzyme-labelled conjugate were both for 2h at 37~ The reaction was stopped 30 min after adding substrate by adding 25 ul 2M H2SO 4 Fluorescence was measured at an emission wavelength of 492 nm using a fluorescence spectrophometer. The extinction value(A49~)was 0.1. Samples with smaller values were considered virus-free (Wang & ,gang 1991b).

67 Table I a. The morphogenetic responses of 0.3-0.5mm garlic stem-tip cultured on M1-M8 media after 18-day and 50-day of incubation. Media 18-day M1 M2 M3 M4 M5 M6 M7 M8
a

Morphogenetic responses 50-day Callus formation A 7cm shoot with 6-7 micro-shoots Leaf primordium curling A 3cm shoot with 4 micro-shoots A 4cm shoot with 4 micro-shoots A 2cm shoot with 2 micro-shoots A 3cm shoot with 2 micro-shoots A 7cm shoot

Turgid growth of the base Leaf primordium elongation, protuberances on the base Swollen growth of the base Turgid growth of the base, leaf perimordium elongation Leaf primordium elongation Turgid growth of the base Turgid growth of the base Leaf primordium elongation

30 stem tips were tested in each medium.

Table 2. ELISA test results for tube plants derived from stem-tip and scape-tip. Stem-tip >0.Smm No. of plantlets tested No. of virus-free plantlets Vtrus elimination rate(%) 117 31 26.5 0.3-0.Smm 205 93 45.4 Scape-tip 2.0-3.0mm 156 121 77.6

Fig. A: Scape tips were cultured on M2 medium. Fig. B: Micro-shoots were produced from a scape tip after 30-40 days culture. Fig. C: Roots and bulblets were formed from 90-day old scape tips.

68 medium only after 30-40 days culture(Fig. B). Also, the growth of shoots from scape-tip was far better than that from stem tips. Micro-shoots were seperated and transfered from the 50-day old stem-tip and 35-day old scape-tip to a hormone-flee MS medium. Roots were produced from the micro-shoots after transftering 2-week on the hormone-free MS medium. It was highly easy to produce roots from garlic calli and shoots(Wang et al. 1992, 1994). Finally, bulblets(diameter 5-6 mm) were formed from the bases of shoots in 150-170 days old stem tips and 90-120 days old scape tips(Fig. C). 50 mg leaves were taken in aseptic condition from each tube-plant for the ELISA test. The ELISA test data are showed in Table 2. The ELISA test depends on the fact that antibodies or antigen can be linked to an enzyme, with the complex relating both immunological and enzymatic activity. The ELISA provides a more accurate, rapid and sensetive method for virus detection(Voller et al. 1979). In previous reports, the electron microscopic method(Abo El-Nil 1977) and the method of indicated plant infectivity (Sant 1980) were used in the virus detection. The electron microscopic method is timeconsuming and the method of indicated plant infectivity is insensetive and inaccurate. The virus elimination rate from stem tips with 1-2 leaf primordia was higher than the rate from stem tips with 3 leaf primordia. This means that the bigger the stem-tip, the greater the number of virus particles in the explants. The virus elimilation rate from scape-tip was much higher than that from stem-tip(0.3-0.5 ram, >0.5 mm). The period of plantlet formation from scape-tip culture was far shorter than that from stem-tip culture. It was concluded that scape-tip culture provided an effective procedure for garlic virus elimination. Until now, there have been few reports of plant scape-tip culture. The idea of garlic scape-tip culture is based on the fact that plant seed embryos are virus-free. This suggested that there is a mechanism of self-eliminating virues in the growth of garlic scape-tip and the development of plant seed embryos. The present procedures of the garlic scape-tip culture and the ELISA test offer an effective strategy for eliminating viruses from the vegetive propagated species. gratefully acknowledged.

References
Abo El-Nil (1977) Plant Science Letters 9:259-264. Clark MF & Adames AN (1977) J. Gen. Virol. 34:475783 He JY, Wang HL, Wang TK, Ma Y (1990) J. TAFST. (1):17-20. Ma Y, Wang TK, Wang HL, He JY, Guo CJ (1991) J. Tianjin Norm. Univ. (1):70-75. MohamedNA & Young BR (1981) Ann. Appl. Biol. 97:65-74 NagakuboT, Nagasawa A, Ohkawa H (1993) PI. Cel. Tis. Org. Cult. 32:175-183. Sant S (1980) Scientia Horticulturae 13:47-52. VoUerA, Bidwell DE, Bartlett A (1979) A guide with abstracts of microplate application, p l-125, Flowline Press, Guernsey. Wang P, Huang L (1974) J. Chin. Soc. Hort. Sci. 20:7987. Wang HL, He JY, Ma Y, Hong RY, Wang TK (1991a) Act. Agri. Boreali-Sin. 6(1):74-80. Wang HL, Kang YQ (1991b) J. Biol. 6(2):29-30. Wang HL, Kang YQ, Zhang CJ, Ma Y, Wang TK (1992) Act. Agri. Boreali-Sin.7(3):66-70. Wang HL, Kang YQ, Zhang CJ (1994) Act. Agri. Boreali-Sin. 9(1), in press.

Acknowledgements. The authors are grateful to Dr. J.


D. Thompson at the Center of Plant Evolution and Ecology, CNRS, France, for assistance in revising the English of the work. The financial support from the TCAS, Goverment of the People's Republic of China is