Plant Cell Reports

Plant Cell Reports (1995) 15:17-21

© Springer-Verlag1995

Regeneration of whole plants from protoplasts isolated from tissue-cultured shoot primordia of garlic (Allium sativum L.) Masanori Ayabe 1, Kenji Taniguchi 2 and Shin-ichiro Sumi 1 1 Institute for Biotechnology Research, Wakunaga Pharmaceutical Co., Ltd., 1624 Shimokotachi, Koda-Cho, Takata-Gun, Hiroshima 739-11, Japan z Laboratory of Plant Chromosome and Gene Stock, Faculty of Science, Hiroshima University, 1-3 Kagamiyama, Higashi-Hiroshima City, Hiroshima 724, Japan Received 14 December 1994/Revised version received 11 April 1995 - Communicated by A. Komamine

Abstract

Protoplasts derived from tissue-cultured shoot primordia of garlic (Allium sativum L.) initiated successive cell divisions within 4 days and formed small individual calli (0.2mm in diameter) after 5 weeks of culture on Gamborg's B5 medium supplemented with 0.1% casein hydrolysate, lmg/1 1-naphthaleneacetic acid and lmg/1 6-benzylaminopurine. Plating efficiency was roughly 5% at the density of lxl04 protoplasts/ml of medium. Adventitious buds developed from the calli during subsequent subculture on Gamborg s B5 medium supplemented with 40mg/1 adenine and 10% coconut milk. When transferred to the same medium without supplements, these buds grew into shoots and rooted. The regenerated garlic plantlets were successfully transferred to the greenhouse and grew into whole plants. K e y w o r d s : garlic, Allium sativum L., shoot primordia, protoplasts, plant regeneration.

Introduction

Garlic (Allium sativum L.) is an important culinary plant as well as a medicinal herb due to its ability to improve the taste of food and to its antibiotic, antitumor and antithrombic effects in animal cells (Fujiwara and Natata 1967). Because garlic is sterile, crop improvement by means of cross fertilization is precluded. Somatic hybridization or DNA transfection through protoplasts may provide a way to overcome this. Of the very few reports on protoplast culture of the genus Allium, most deal with A.cepa and Correspondence to: S. Sumi

A.fistulosum, not with A.sativum (Wang et al. 1986; Balakrishnamurthy et al. 1990; Fellner 1991). In spite of intensive studies done in several laboratories, only a few instances of cell division have been reported in cultured protoplasts of A.sativum (Opateny and Havranek 1977; Fogher and Corti 1982; Nishio et al. 1989). Only Oosawa and Takayanagi (1984) reported success in inducing garlic protoplasts prepared from leaves to divide several times in the two Japanese cultivars, "White roppen" and "Ishu-wase". The earlier results for protoplast cultures of certain cereal plants were similarly poor, but lack of cell division later was overcome by the use of selected cell lines that differentiate into whole plants (Potrykus et al. 1979; Vasil and Vasil 1980; Lu et al. 1981; Vasil et al. 1983). Tanaka et al. (1983) induced a unique domeshaped tissue, which they called the "shoot primordium", from Haplopappus gracilis and showed that it has marked potential for regeneration into whole plants that have remarkable genetic stability. Subsequent reports on the shoot primordia of various plants are many, and this special tissue has been shown to be a good source of micropropagation, as well maintaining the genetic stock (Miyagawa et al. 1986; Tanaka et al. 1988; Kondo et al. 1991; Takano et al. 1991). In addition, Ito et al. (1987) succeeded in regenerating whole plants from protoplasts isolated from the shoot primordium of

Populus. The properties shown by these shoot primordia suggest that the shoot primordium may be a good source of garlic protoplasts. Accordingly, we first induced shoot primordium from garlic shoot apex and used it to prepare protoplasts from which we could regenerate whole garlic plants.

18 Materials and Methods

Results

Plant material Garlic (Allium sativum L cv. White roppen) cultivated

Induction of shoot primordia

on our farm in Hokkaido, Japan was used in all the experiments.

Induction of shoot primordia Shoot primordia were induced from garlic shoot apex according to the method of Tanaka and Ikeda (1983). Garlic cloves were cut into small cubes and sterilized for 5rain in 70% ethanol. After removing both the scale and foliage leaves, each shoot apex (about 0.5ram in diameter) was excised and placed in a test tube (30ram x 200ram) containing 20ml of modified Linsmaier-Skoog (LS) medium (Linsmaier and Skoog 1965) supplemented with 1naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). Cultures were incubated at 25°C on rotary stages rotating at 2 cycles/rain under 50001ux continuous fluorescent illumination. The shoot primordia induced were subcultured for propagation at intervals of 2 weeks.

Protoplast isolation Four types Of garlic source material were used for protoplast isolation: immature etiolated leaves from cloves, green leaves, shoot primordia and calli derived from shoot apices in LS media supplemented with 2mg/1 BAP according to Oosawa et al. (1981). One gram of each source was cut into small pieces and treated with 10ml enzyme solution containing 1% Cellulase Onozuka RS (Yakult Honsha Co., Tokyo Japan), 0.1% Pectolyase Y23 (Seishin Pharm. Co., Tokyo Japan), 0.6M mannitol and 10mM CaC12 under shaking at 60rpm for 5 to 6hr at 28°C. The protoplasts obtained were filtered through 40~tm nylon mesh then centrifuged at 800rpm for 5min. After being washed 3 times with 0.6M mannitol solution containing 10mM CaC12, they were purified by centrifugation on 20% sucrose at 1000rpm for 10min.

Plant regeneration The calli derived from the protoplasts were transferred to B5 medium supplemented with lmg/l NAA, lmg/l BAP, 40mg/l adenine and 10% coconut milk then solidified with 1.5% agarose. After 3 weeks, several adventitious buds developed. Rooting of the shoots was induced by transfer to the same medium without growth regulators.

Discussion

We first tried to induce garlic shoot primordia by the technique of Tanaka and Ikeda (1983). Shoot apices excised from garlic cloves were cultured in liquid LS media containing N A A and B A P in combinations of 0, 0.02, 0.2, 2.0 and 4.0mg/1 of each h o r m o n e . After 2 to 3 m o n t h s , different types o f tissues were p r o d u c e d d e p e n d i n g on the N A A c o n c e n t r a t i o n (Table 1). At low N A A concentrations (0 and 0.02mg/1), a shoot was induced exclusively from the shoot apex and grew without rooting. At a moderate NAA concentration (0.2mg/1), calli, as well as "green tissue" which was anomalously swollen, formed. The high N A A concentrations of 2.0 and 4.0mg/1 inhibited both the growth and d e v e l o p m e n t of the shoot apices. The effect o f B A P w a s m u c h less marked than that o f NAA, shoots being induced at all the concentrations from 0 to 4.0mg/1 (Table 1). No shoot p r i m o r d i u m , h o w e v e r , f o r m e d under any of these conditions. Table 1 Induction of shoot primordia from shoot apices in LS media containing NAA and BAP NAb. (rag/l)

o

0.02

BAY(rag/l) 0.2

S(1/4; N(3/4)

S(2/4) N(2/4)

S(3/4) N(1/4)

0.02

S(2/4) N(2/4)

s (2/4) N(2/4)

0.2

c(314) N(1/4)

2.0

4.0

Protoplast culture To examine the plating efficiency, isolated protoplasts were resuspended at final concentrations of 0.2 - 10.0 x 105 protoplasts/ml in 10ml of Gamborg's B5 (B5) media (Gamborg et al. 1968) supplemented with lmg/l NAA, lmg/l BAP, 0.5M mannitol and 0.1% casein hydrolysate. This suspension was mixed in petri dishes with an equal volume of culture medium containing molten 1.2%(W/V) low-melting-point agarose, after which the dishes were cultured at 25°C in the dark. The microcalli that formed after about 5 weeks were transferred to solid B5 medium without mannitol and culture continued.

and

2.0 s (3/4)

4.0

N (1/4)

S (4/4)

S(2/4) N(2/4)

S(2/4) G(U4) N0/4)

s (1/4) N(3/4)

G(2/4) N(2/4)

G(2/4) N(2/4)

G(2/4) N(2/4)

G (4/4)

N(4/4)

N(4/4)

N(4/4)

G(U4) N(3/4)

o0/4) N (3/4)

N(4/4)

N(4/4)

N(4/4)

N(4/4)

N(4/4)

a S; shoot~ G; anomalouslyswollengreen tissue, C; ealli, N; no growth b (numberof shoot apices developed/ number of shoot apices planted)

We therefore investigated various concentrations o f the LS basal m e d i a and s u c r o s e for their efficacy in inducing shoot primordia. We prepared t w o and four fold d i l u t e d LS basal m e d i a s u p p l e m e n t e d with N A A and B A P in the same series of combinations described above. After all the trials, half-strength LS m e d i u m containing 1%

19 sucrose was shown to be optimal. After 3 months of culture, masses that constituted of many small globular tissues had been produced from one of every four explants cultured under two conditions: 0.02mg/l NAA and 0.2mg/l BAP or 0.2mg/l NAA and 2.0mg/l BAP (Fig.I-A,B and Table 2). This shoot primordium-like tissue developed from the basal region of shoots grown initially from shoot apices. The tissue was transferred to the same media and subcultured for propagation. The tissue propagated more rapidly and stably in medium supplemented with 0.02mg/1 NAA and 0.2mg/1 BAP than under the other conditions. The shoot primordium-like tissue doubled in size within 2 weeks. We next examined the various properties of the shoot primordium-like tissue in detail. Microscopic observation of cross-sections of the tissue revealed it was composed of small cells with rich cytoplasm, as commonly are seen in shoot apices (Fig. l-C). The genetic stability of the shoot primordium-like tissue was estimated by analyzing the chromosome numbers of its cells. None of the 100 cells examined showed chromosomal aberrations, the usual diploid chromosome number of 16 being confirmed in all of them. As these observations are consistent with the characteristics of shoot primordia induced in other plant species (Tanaka and Ikeda 1983; Miyagawa et al. 1986; Tanaka et al. 1988; Kondo et al. 1991; Takano et al. 1991), we concluded that the tissues obtained were shoot primordia. T a b l e 2 Induction of shoot primordia from shoot apices in half-strength LS media (sucrose 1%) containing N A A and BAP

BAr'(mg~)

NAA

(me,a)

0

0.02

0.2

2.0

Sa(2/4; N (2/4)

S (1/4) C (3/4)

S (2/4) N (2/4)

S (2/4) G (1/4) N (1/4)

S (1/4) N (3/4)

S (2/4) N (2/4)

SP(1/4) G (2/4) N (1/4)

G (I/4) N (3/4)

0.2

S (2/4) N (2/4)

S (3/4) C (1/4)

S (2/4) G (1/4) N (1/4)

SP(I/4) S 0/4)

2.0

C (1/4) N (3/4)

G (1/4) N (3/4)

N (4/4)

C (2/4) N (2/4)

0.02

. S; shoot, C; calli, G; anomalously swollen green tissue, SP; shoot pfimordia, N; no growth b (number of shoot apices developed / number of shoot apices planted)

Fig.1 Garlic shoot primordia. A, B: Shoot primordia derived from a shoot apex. (Bar=lcm in A and 0.5mm in B) C: Longitudinal section of shoot primordia. (Bar=0.2mm) D: Plantlets regenerated from shoot primordia. (Bar=lcm)

We investigated the regeneration of plantlets from these shoot primordia and observed highly efficient formation of adventitious buds in solid two fold-diluted LS medium supplemented with 0.02mg/l BAP and 1% sucrose. The adventitious buds were harvested periodically and transferred to fresh medium, which enabled them to develop into plantlets (Fig. l-D).

Protoplast culture In addition to shoot primordia, we used young green leaves, immature etiolated leaves and calli obtained from the in vitro culture of shoot apices as sources of protoplasts. The protoplasts yields were almost the same, 104 to 105 protoplasts per gram of tissue being obtained from all but the young green leaves, for which the yield was noticeably lower. Interestingly, protoplasts from different sources developed different morphologies. Those obtained from shoot primordia that had cells with rich cytoplasm were 20 to 30 rim in diameter (Fig. 2-A) and were more uniform and compact than those from the other sources. We therefore used the protoplasts from shoot primordia in our subsequent experiments. We next investigated various culture conditions to determine the optimum conditions for protoplast growth and development. We examined the effects of natural products other than phytohormones on protoplast culture, using supplements of casein hydrolysate and coconut milk which are known to promote the growth and

20 development of plant cells (Maheshwari et al. 1986). With no natural product supplement, garlic protoplasts divided several times and formed small colonies which did not develop further. In contrast, an addition of casein hydrolysate markedly promoted protoplast cell division and the development of colonies. We therefore used B5 medium supplemented with lmg/l NAA, lmg/1 BAP and 0.1% casein hydrolysate for protoplast culture. On this medium, protoplasts quickly reformed their cell walls and underwent successive cell divisions after 4 days, small colonies appearing after 14 days of culture (Fig.2B,C). These colonies continued to grow, forming microcalli after 5 weeks (Fig.2-D). Visible calli that appeared after 8 weeks (Fig.2-E) were transferred to solidified B5 medium without mannitol for further propagation. The effect of protoplast density on callus formation was examined by changing the final protoplast concentration in the agarose from 104/ml to 5x105/ml (Table 3). Increasing the density tended to inhibit callus formation, the best result being obtained at a density of 104/ml. The plating efficiency was estimated to be approximately 5% under this condition.

Fig.2 Culture of protoplasts isolated from garlic shoot pdmordia. A: Protoplasts freshly isolated from the shoot primordia. (Bar=10ltm) B: First cell division after 4 days of culture. (Bar=101xm) C: A cell colony after 2 weeks of culture. (Bar=10~tm) D: A microcallus after 5 weeks of culture. (Bar=50~tm) E: Visible calli after 8 weeks of culture. (Bar=5mm)

Table 3 Effect of protoplast density on callus formation from protoplasts isolated from garlic shoot primordia

density (No. of protoplasts/ml)

efficiency (%)

0.1x10 s

5.0 -i-0.39

0.2x10 s

3.0

0.5X10 s

1.4 _0.13

1.0xlOS

0.13_+0.024

5.0X10 s

0 . 0 4 ± 0.005

_

_

_

1.23

a Efficiency:%( +__standarddeviation)of protoplaststhatdevelopedinto microcalll.

Plant regeneration Several basal media, MS (Murashige and Skoog 1962), LS, B5 and SH (Shenk and Hindebrandt 1972), supplemented with auxin and cytokinin in various combinations and concentrations were examined for their effects on plant regeneration from protoplast-derived calli. B5 media gave the best result for the propagation of calli, but none of these media promoted regeneration. Some natural products are known to promote regeneration from calli. Skoog and Tsui (1951) found that a supplement of adenine in the medium significantly promoted plant regeneration from tobacco callus. Recently Cid et al. (1994) reported that an addition of adenine was effective for the regeneration of plantlets from calli derived from suspension cultures of garlic. We therefore examined the effects of three natural products: casein hydrolysate, coconut milk and yeast extract, together with adenine on the regeneration of calli derived from garlic protoplasts. Additions of both adenine and coconut milk had dramatic synergistic effects on the regeneration of garlic calli. Many adventitious buds formed on the calli, the highest efficiency (75%) occurring with 40mg/1 adenine and 10% coconut milk in B5 media containing lmg/l NAA and BAP (Fig.3-A). The adventitious buds that formed grew into shoots that rooted when placed on supplementfree B5 media, but with considerably lower efficiency (nearly 1%) than there was for adventitious bud formation. The regenerated garlic plants were transferred successfully to the greenhouse and grew normally as shown in Fig.3-B,C,D.

21

References

Fig.3 Plant regeneration from protoplast-derived garlic calli. A: Adventitious buds regenerated from the protoplastderived calli. B: Multiple shoots derived from the adventitious buds. C: Rooted plantlets. D: A protoplast-derived garlic plant potted in vermiculite. (Bar=-icm)

In conclusion, we have succeeded in regenerating whole garlic plants from protoplasts via callus using shoot primordia as the starting material for protoplast isolation. Our results appear to be attributable to the high viability and high capacity for regeneration of garlic shoot primordia, as has been reported for the shoot primordia of many other plant species. The possibility of generating new garlic lines by means of genetic engineering and cell fusion as well as by somaclonal variation is indicated.

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