Pivotal roles of picloram and gelrite in banana callus culture

Environmentaland ExperimentalBotany, Vol. 28, No. 3, pp. 249-258, 1988.

0098-8472188 $3.00 + 0.00 © 1988. Pergamon Press pie

Printed in Great Britain.

P I V O T A L R O L E S OF P I C L O R A M A N D G E L R I T E IN B A N A N A CALLUS C U L T U R E LI-CHUN HUANG*

and DAUH-LIAN

CHI

Institute of Botany, Academia Sinica, Nankang, Taipei, Taiwan

(Received 23 September 1987; acceptedin revisedform 18 March 1988) HUANG L.-C. and CHI D.-L. Pivotal roles of picloram and gelrite in banana callus culture. ENVmONMENTAL AND EXPERIMENTAL BOTANY 28, 249--258, 1988. Vigorous development of callus was achieved only when the nutrient medium was supplemented with picloram and solidified with gelrite. Gelrite also eliminated tissue and medium discoloration. The callus resulted from intensified activity of the primary thickening meristem of shoot-tip explants. After continuous culture for 1.5 years, only a small proportion of the callus cells displayed the normal chromosome number, 3N = 33, the majority being variable aneuploids. Liquid suspension cultures were established by transferring the relatively friable callus to a constantly shaken liquid medium.

Key words: Musa, banana, callus, cell suspension, gelrite, picloram.

INTRODUCTION

TISSUE culturing has been established as an effective method of reproducing clonal banana propagules in commercial quantities (unpublished information, Oglesby Plant Laboratory, Florida). Its utility as a parasexual alternative to traditional breeding methods of varietal improvement, e.g. source ofsomaclonal variants, hybrids and cybrids by protoplast fusion, etc. is currently under exploration. Callus establishment and plant regeneration have been reported previously, e.g. CRONAUER and KRIKORIAN ~4' and MOHAN RAM and STEWARDJ8~ The present data base tbr tissue culture lacks general information about banana species and cultivars, largely because commonly employed growth substances are relatively ineffective and severe tissue and nutrient medium discoloration and deterioration occur. Both problems may have been resolved by this investigation.

* To whom correspondence should be addressed.

MATERIALS

AND METHODS

Shoot tips, 2-3 m m tall and including two-four small emerging leaves and obtained from a stock of banana (Musa sapientum L.) tissue culture that proliferated continuously as shoots (Fig. 1), were used as explants. The stock was kindly provided by the Taiwan Banana Research Institute, Tainan. Basal nutrient medium ingredients were MURASHIGE and S K O O G (9) salts; 3% sucrose; and, in mg/l: additional phosphate as NaH2PO4 • H~O, 170; adenine sulfate'2H20, 50; i-inositol, 100; thiamine. HC1, 1; pyridoxine- HC1, 0.5; nicotinic acid, 0.5; and glycine, 2. The auxins IAA (3indoleacetic acid), IBA (3-indolebutyric acid), NAA (l-naphthaleneacetic acid), 2,4-D (2,4dichlorophenoxyacetic acid) and picloram (4amino-3,5,6-trichloropicolinic acid) were tested in concentrations ranging ti'om 1.5× 10 4 to 1.5 x 10-1 m M for callus-initiation effects, and 2,4-D and picloram were examined further with subcultured callus. Similarly, the cytokinin 2iP (N6-isopentyladenine), BA (N6-benzyladenine) and kinetin (N6-furfuryladenine) were examined 249

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L.-C. HUANG and D.-L. CHI

in levels of 2 x 10 -4 to 5 x 10 -2 m M for callus- weight of sedimented cells were made after 3 initiating effects, but only 2iP being evaluated in weeks. All experimental data were evaluated by calsubcultures. Importance of the basal constituents adenine sulfate" 2H20 and NaH2PO4" H 2 0 was culating standard errors of means, following determined. The sugar requirement was studied SNEDECOR. (12) To establish the origin of callus in shoot-apex by comparing sucrose, glucose, fructose and galactose, each at 1.5-9% rates in 1.5% explants, five primary cultures were sampled increments. The choice of gelling agent was estab- every 3 days, fixed in FAA (37% forlished by comparing K C Biological T C agar, maldehyde:glacial acetic a c i d : 9 5 % ethanDifco Bacto-agar, Difco Bacto-purified agar, and ol:distilled water, 2:1 : 10:7, v : v : v : v ) , dehyKelco gelrite. Agar samples were employed at drated through the ethanol series into xylene, and 0.8% and gelrite at 0.2%. The p H of all media embedded in paraplast. Longitudinal sections, 10 was set at 5.7 with 1 N K O H or HCI, just prior #m thick, were prepared and stained with safto their final dilution and gelling-agent addition. ranin-fast green. The histological procedure comThe media were dispensed in 25-ml quantities bined selected steps from BERLYN and MIKSCHE,(1) into 25-x 150-ram glass culture tubes, and the JOHANSEN(5) and SASS.(1~) Chromosome counts of callus cells were pertubes capped with Bellco kaputs and autoclaved at 1.05 kg cm -2 for 5 rain. All test substances formed according to an unpublished method of were included in media prior to autoclaving. At Professor W. B. Storey of the University of Calileast 10 and up to 15 cultures were employed per fornia, Riverside. Roots, 1 cm long and obtained by rooting explant donor shoots, and callus, 10 treatment. The cultures with one explant each were incu- mg quantities from tissues 5-7 days after transfer bated in continuous darkness at 27°C. Obser- to fresh medium were immersed in IPC (isovations of their development were made after 4 propyl-N-(3-chlorophenyl)carbanilate, 10 mg/1) solution for 90 rain, fixed in propanoic acidweeks. Stock cultures of callus were established by sub- alcohol (100 ml propanoic acid and 200 ml 95% culturing lO0-mg portions every 4 weeks in freshly ethanol) for 20 rain, and stored in 70% ethanol. prepared medium of progressively improved com- Following maceration in 10% HC1 for 20 min, position. For experiments, lO0-mg portions of the tissues were washed with warm water, dipped stock callus were used and observations and fresh in 45% propanoic acid, stained with lacto-propanoyl-orcein (a quantity of 0.5-0.8 g orcein was weight measurements were made after 4 weeks. dissolved in 45 ml boiling propanoic acid and Liquid suspension cultures were established by boiled for a few min, then diluted with 35 ml transferring the callus to a medium of the same distilled water; after cooling, 5-20 ml lactic acid composition, but without gelling agent. The soluwas added; the final solution was refrigerated and tion, also in 25-ml quantities, was dispensed into 125-ml Erlenmeyer flasks. Echopla stoppers filtered), squashed, and examined. Only metaphase figures in polar view were counted. (Microwave Enterprises, Inc., Taipei), designed to facilitate gas exchange while excluding airborne infectious agents, were used for flask closures. The RESULTS suspension cultures were also incubated at 27°C, but under 16-hr daily exposure to 4.5 nEcm -2 The auxins IAA, IBA and NAA were totally sec -1 illumination from Toshiba FL-40SBR/38 ineffective in causing callus development. At best, fluorescent lamps. The flasks were swirled conthey stimulated rooting of shoot-apex explants. 2,4-D suppressed rooting and elicited a very mild tinuously at 150 rpm on a Hotech orbital shaker. callus response (Fig. 2). Substantial callus develStock cultures of liquid suspension were mainopment occurred only when nutrient media contained by transferring 500-mg quantities of sedimented cells (270 g, 10 min) to fresh nutrient tained picloram. Its optimum concentration was 5 x 10 3 m M (Fig. 3). Considerable callus forsolution at 3-week intervals. Suspension culture experiments also employed 500-mg quantities of mation also occurred in the medium with cells and measurements of volume and fresh 1.5x 10 .3 m M picloram, but only roots

PIVOTAL ROLES OF PICLORAM AND GELRITE

FIG. 1. Sample of Musa sapientum tissue culture that proliferated continuously as shoots, and from which were obtained the shoot-tip explants.

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L.-C. H U A N G and D.-L. CHI

FIo. 2. Weak callus-generating effect of 2,4-D on excised b a n a n a shoot tips. Left to right: 0, 15, 50, 150 and 500 x 10 -4 m M 2,4-D 50 m M picloram reference. FIo. 3. Strong callus-generating effect of picloram on excised b a n a n a shoot-tips. Left to right: 0, 1.5, 5, 15, 50 and 5 0 0 × 1 0 '~mM.

PIVOTAL ROLES OF PICLORAM AND GELRITE

FIG. 5. Prevention of discoloration and promotion of banana callus growth by gelrite. Left: 0.8% TC agar, right: 0.2% gelrite.

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FIG. 9. Origin of callus and roots in banana shoot-tip explants. All 72 x . (A) Longitudinal section of initial explant. The shoot tip after 3 (B), 6 (C) and 9 (D) days in culture. Arrows indicate primary thickening meristem activity and generated callus. Development of roots (arrows) is shown in (E) and of leaf callus (arrows) in (F).

PIVOTAL ROLES OF PICLORAM AND GELRITE

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FIo. 4. Comparative yields ofsubcuhured banana callus in media containing 2,4-D and picloram. Error bars = standard error of means.

developed at levels of 5 x 10 4 m M and lower. Concentrations of 1.5 x 10 _2 and 5 x 10 -2 m M suppressed callus development. Callus subcultures in the presence of picloram or 2,4-D displayed growth promotion. However, the m a x i m u m callus yield with 2,4-D (optimum concentration, 5 x 10 4 mM) was only half that obtained with picloram (Fig. 4). The optimum level of picloram for growth promotion of subcultured callus ranged between 1.5 and 5 x 10 -3 mM, the latter was selected as "standard". The cytokinins 2iP, BA and kinetin mainly intensified blackening of excised shoot apices and resultant callus and repressed growth of callus. The severity of discoloration was directly related to the cytokinin concentration. Cytokinins were thus excluded from further use as banana callus supplements. As a carbon source, sucrose was the most effective, followed by glucose, in promoting growth of banana callus. Sucrose and glucose produced optimum growth results at a concentration of 1.5%, with sucrose yielding 50% more callus than glucose. However, tissue discoloration was more pronounced at the 1.5 than the 3% level. Galacrose was toxic at all concentrations. Fructose was not toxic, but totally ineffective in supporting callus growth. The conspicuous feature of banana tissue cultures has been their intense discoloration, of both tissue and medium. The discoloration is usually

255

associated with growth inhibition and frequently with tissue death. Addenda of activated charcoal powder did not reduce the discoloration or the repression of callus growth. This problem was resolved by simply replacing the agar with gelrite/7) as the gelling agent. The prevention of blackening by gelrite, but not by agar, can be seen in Fig. 5. Similarly, vigorous growth of banana callus on media solidified with gelrite can be observed in the data of Fig. 6. Adenine sulfate and NaH2PO4 were unnecessary for banana callus culture. Adenine sulfate suppressed callus growth, the yields being 0.8-t-0.2, 0.9-t-0.1, 0.4-t-0.2, 0.2+0.1 and 0.2+0.1 g tissue, respectively, for 0, 25, 50, 75 and 100 mg/1 of the supplement. Exclusion of N a H 2 P O 4 • H 2 0 did not diminish callus growth. Exposure of banana callus cultures to 4.5 nEcm -2 sec l illumination, 16 hr daily, using Toshiba FL-40SBR/38 lamps, reduced callus yield to onequarter that of dark-grown tissues. The yield in darkness averaged 2.3___0.39 g, whereas that under illumination was 0.58-t-0.10 g. The growth curve for banana callus, obtained in darkness and with the nutrient medium properly adjusted, can be seen in Fig. 7. Callus yields increased very slowly over the first 4 weeks, and rose exponentially thereafter. The final fresh weight after 8 weeks was 70 times that of the inoculum callus. However, the tissue after 8 weeks had lost its original whitish color and had started to brown, signalling time for removal to fresh medium. Chromosome counts of callus cells after 1.5 yr of continuous culture disclosed only 13% (six of 45 mitotic figures) with the normal number of 3N = 33. The remainder displayed variable aneuploidy, 22% (10 of 45) of which were below and 65% (29 of 45) of which were above the triploid level. The lower numbers ranged from 8 to 30 and the higher numbers, from 36 to 108. Extensive morphogenetic experiments were not performed with the established callus. Combinations of several levels of picloram and BA did not result in rooting or shoot formation, in illuminated and unilluminated cultures. When transferred to liquid medium and shaken continuously, the relatively moist and friable banana callus produced a fine suspension, comprised largely by few-celled aggregates and by a

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L.-C. HUANG and D.-L. CHI

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Fro. 6. Yields of subcultured banana callus in media with different gelling agents. All agar preparations were included in a 0.8% concentration, and gelrite in 0.2%. Error bars denote standard error of mean.

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FIO. 7. Growth curve of subcultured banana callus in picloram gelrite medium. Error bars indicate standard error of means.

smaller fraction of free cells. T h e typical growth curve of an established suspension culture can be seen in Fig. 8. Preliminary experiments revealed that the suspension culture served effectively as a source of b a n a n a protoplasts, although viability of the protoplasts was not confirmed. T h e callus of b a n a n a shoot-apex explants appeared to arise by intensified cell division in their primary thickening meristem. T h e intensified activity was first evident in cultures sampled after 3 days (Fig. 9B). After 6 days, the new cells had caused considerable swelling and rupturing of tissues in the subapical flanks (Fig. 9C). T h e callus-forming process thereafter was simply one of further swelling and rupturing of tissues, eventually extending down the length of the explant (Fig. 9D). Adventitious roots differentiated, particularly from cells at the basal cut surface of the explant (Fig. 9E). Callus formation was also

PIVOTAL ROLES OF PICLORAM AND GELRITE observed in emerging leaves, among cells surrounding the midrib (Fig. 9F). This tissue, however, did not contribute significantly to the ultimately established banana callus culture. DISCUSSION

MOHAM RAM and STEWARD (s) w e r e the first to attain banana callus in culture. They achieved their success by employing pulp tissue excised from pre-climacteric fruits and a nutrient mcdiurn supplemented with the auxin, 2,3,6-trichlorophenylacetic acid (2,3,6-TPA), and an undefined natural complex, coconut milk. 2,4-D was weakly effective and IAA totally ineffective in stimulating callus formation. In the best instance, a 10-fold increase of tissue was obtained after 20 days. The authors noted that the capacity for callus development varied with the genotype. Greatest success thus far has been attained by CRONAUER and KRIKORIAN.(4) Vigorously growing callus and cell suspension cultures, capable of manifesting somatic cell embryogenesis, were established from shoot-apex explants of the plan-. tain, Musa ABB, a hybrid between M. acuminata (AA) and M. Balbisiana (BB). These authors employed a medium containing 2,4-D and 2,4,5-T (2,4,5-trichlorophenoxyacetic acid). They apparently encountered no major difficulties. Experience has clearly shown, however, that an exceptionally potent auxin must be provided. Excised banana tissues also manifest rapid and very severe discoloration, eventually producing an intensely black medium as well as tissues. Both problems have been resolved in this investigation with M. sapientum. Picloram serves as a potent auxin and gelrite, a gelling agent, overcomes the problem of tissue and medium discoloration. The strong auxin effects of picloram were first observed by KEFFORD and CASO. (b) Picloram was described as being active in a variety of auxin bioassays, including induction of cell division in tobacco pith explants, in extremely low concentrations. Subsequently, CHERNOVAet al. 12)discovered that picloram was more effective than 2,4-D in generating callus in wheat explants. A more extensive study by COLLINSet alJ a~disclosed that picloram was consistently superior to 2,4-D, NAA and IAA, requiring a considerably lower concentration for callus initiation in alfalfa,

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clover, corn, Jack bean, soybean, tobacco and wheat. The superiority of picloram over other auxins has now been extended to onion callus culture./~°/ For banana callus, its growth promoting effect is also unequalled by 2,4-D, NAA, IBA and IAA. Its optimum concentration is near 1.5 x 10 -s mM. Picloram is clearly more potent than all other readily available auxins for tissue cultures of a multitude of species. It deserves trial whenever the question of auxin effectiveness arises. The basis for its high potency remains unelucidated. The tissue discoloration of banana callus culture has been avoided in this investigation by employing gelrite for gelling agent. The basis of this unexpected advantage of gelrite over agar also remains unelucidated. More important, the extent of more general applicability of the observation in overcoming discoloration problems in plant tissue cultures, including those of other Musa species and cultivars, deserves further study. The principal source of callus in banana shootapex explants is the primary thickening meristem. Like most other monocotyledonous plants, this meristem is the basis for lateral enlargement of banana stems. Competence of the callus from intensified activity of the primary thickening meristem to manifest organogenesis has not been established, although preliminary experiments have not been encouraging. Also, maintenance of the callus through subcultures eventually results in a population of predominantly variable aneuploid cells. This genetic change could cause a reduction of morphogenetic competence. Banana callus generated by the present method can be further cultured as cells in liquid suspension. The latter can then serve as sources of protoplasts. Acknowledgements The authors thank Professor T.

Murashige, University of California, Riverside, for assistance in preparing the manuscript. REFERENCES

1. BERLYNG. P. and MIKSCHEJ. P. (1976) Botanical Mitrotechnique and Cytochemistry. Iowa State University Press, Ames, 2. CHERNOVAL. K., PROKHOROVM. N. and FILXNKOLBAKOVB. V. (1975) Comparison of the dedifferentiating effects of 2,4-D and 4-amino-

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4.

5. 6.

7.

L.-C. H U A N G and D.-L. CHI 3,5,6-trichloropicolinic acid on tissues of legumes and cereals. Fiz. Rast. 22, 170-175. COLLINS G. B., VIAN W. E. and PHILLIPS G. C. (1978) Use of 4-amino-3,5,6-trichloropicolinic acid as an auxin source in plant tissue cultures. Crop. Sci. 18, 286-288. CRONAUER S. and KIRKORIAN A. D. (1983) Somatic embryos from cultured tissues of triploid plantains (Musa 'ABB'). Pl. Cell Rpt. 2, 289-291. JOHANSEND. A. (1940) Plant microtechnique, 1st edn. McGraw-Hill, New York. KEFFORD N. P. and CAso O. H. (1966) A potent auxin with unique chemical structure--4-amino3,5,6-trichloropicolinic acid. Bot. Gaz. 127, 159163. KELCO (1985) Gelrite gellan gum thermal-revers-

8. 9. 10.

11. 12.

ible gelling agent as an agar replacer. Kelco Commercial Development CD-35, San Diego. MOHAN RAta H. Y. and STEWARD F. C. (1964) The induction of growth in explanted tissue of banana fruit. Can. J. Bot. 42, 1559-1579. MURASHIGE T. and SKOOG F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Pl. 15, 473-497. PHILLIPSG. C. and LUTEVN K. J. (1983) Effects of picloram and other auxins on onion tissue cultures. J. Am. Soc. Hort. Sci. 108, 948-953. SASSJ. E. (1958) Botanical microtechnique, 3rd edn. Iowa State College Press, Ames. SNEDECORG. W. (1946) Statistical methods, 4th edn. Iowa State College Press, Ames.