Plant Cell, Tissue and Organ Culture 6:159-166 (1986) © Martinus Ni]hoffPublishers, Dordrecht - Printed in the Netherlands

Short communication

In v i tro p r o p a g a t i o n o f b a n a n a (Musa spp . ) : i n i t i a t i on ,

p r o l i f e r a t i o n a n d d e v e l o p m e n t o f s h o o t - t i p c u l t u r e s o n

d e f i n e d m e d i a

W.C. WONG

Department of Primary Industries Plant Pathology Branch, Meiers Road IndooroopiUy, Queensland, Australia 4068.

(Received 11 September 1985)

Key words: banana, in vitro multiplication, cytokinins

Abstract. In vitro multiplication of banana (Musa spp.) from shoot-tip explants isolated from lateral suckers is described. Using explants with apical domes, a total of 22 banana eultivars were successfully cultured on a modified Murashige and Skoog's medium containing 6-benzylaminopurine (BA) and indolebutyric acid (IBA). Shoot-tip explants could be induced to produce multiple shoot initials in the presense or absence of apical domes, but the survival rates were higher ff apical domes were retained. Cultivars varied widely in their multiplication rates in response to cytokinins, BA being consistently more effective than kinetin (Kn). Although Kn was less effective in this regard, it stimulated vigorous root growth. Rooted plantlets were successfully established in soil.

Introduction

Banana (Musa spp.) is propagated vegetatively. The rate of multiplication of a clone is slow and suckers may be infested with important fungal pathogens, such as Fusarium oxysporum Schlecht. ex Fr. f. sp. cubense (E.F. Smith) Snyd. & Hans., the causal organism of Panama disease [10,12]. In vitro

propagation through shoot-tip culture may overcome these problems. In most cases fungal pathogens are eliminated if aseptic techniques are observed when shoot-tips are cultured. If not, fungal contamination can be detected in culture media. Shoot-tip culture of banana has recently been studied in many laboratories [1, 2, 3, 4, 6, 7, 13, 14]. However, there is still a lack of published data on the survival of shoot-tip explants on initiation medium, and the effect of cytokinins on proliferation and growth of tissue-cultured shoots. This paper reports the survival and growth of shoot-tip explants of 22 banana cultivars on initiation medium. The influences o f cytokinins on shoot proliferation and rooting of shoots are also described.

Materials and methods

Plant materials

Banana suckers from 22 cultivars in six genomic groups showing no virus symptoms were dug from the field. At least two lateral suckers with an

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average height of 15 cm were obtained from each plant. The suckers were stored in the laboratory at room temperature (25 + 2°C) and they were tissue-cultured within two days. All cultivars were cultured in May 1983 except for cv. Calypso (March 1984) and, cv. Hsein-Jen-Chiao and cv. Grand Nain (August 1984). Experiments on shoot multiplication and root growth were conducted between September 1983 and February 1984.

Preparation of explants

Shoot-tip explants of banana were prepared by removing the outer layers of tissue from suckers with a clean knife. Tissue blocks (10 × 15 ram) containing shoot-tips and rhizomatous bases were surface sterilised for 15rain in a solution of sodium hypochlorite containing 1% free available chlorine, and 0.01% Tween 80. The dead tissue of each sterilised block was cut off to leave a 5 × 8 mm portion containing an intact apex and one or more pairs of leaf primodia together with 3 mm of rhizomatous base. This sterilised tissue block was cut longitudinally into two, the half containing the apical dome was retained and used as an explant; the remainder was discarded. The second set of explants from 18 of the 22 cultivars (Table 1) were prepared as above but had their apical domes together with subjacent leaf primodia removed so that the effect of apical dome on shoot initiation could be determined. All explants were placed on initiation medium (IM, see below) and incubated at 25 + I°C with a 16h photoperiod provided by cool white fluorescent tubes at 60 pEru -~ s -1 .

Culture media and experimental detail

The basal medium (BM) contained the inorganic salts of Murashige and Skoog [8] plus glycine (2rag.l-I) , meso-inositol (100rag.1-1), thiamine HC1 (0.5 rag.l-l), pyridoxin-HC1 (0.5rag.1 -~), nicotinic acid (5rag.1 -~) and sucrose (20mg.l-~). The pH was adjusted to 5.7 before addition of Bacto-agar (7g.1-1). The initiation medium (IM) used for induction of shoot-initials from shoot-tip explants consisted of BM supplemented with 5 mg.1 -~ 6- benzylaminopurine (BA) and 0.1 mg.1-1 indole-3-butyric acid (IBA).

The rate of shoot proliferation and root growth of excised shoots were evaluated using BM containing 5 levels (see result sections) of BA or kinetin (gn).

Active and uniform excised shoots were transferred from 1M to BM and maintained for 14 days prior to the different cytokinin treatments. Ten shoots arranged in a glass jar (6 × 10cm) comprised a plot and four replicates were made. All experiments, except those reported in Table 1, were repeated on three occasions. The data on shoot proliferation and root growth were recorded after incubation for six weeks.

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Table 1. Survival and growth of shoot-tip explants from 22 banana cultivars (Musa spp.) after 56 days cultured on initiation medium

Genome Cultivar Shoot pieces with Shoot pieces with Type apical domes intact apical domes removed

Survival* Survival*

AA Sucrier 6/8 NT AAA Gros Michel 4/6 NT AAA Lacatan 8 [8 1 [6 AAA Dwarf Cavendish 8]8 1]8 AAA Robusta 6/6 3/7 AAA Red Dacca 4/6 0[6 AAA Hsein-Jen-Chiao 8/8 1/7 AAA Grand Nain 6/6 NT AAA Mons Mari 7/7 2/8 AAA Williams 12/12 3/15 AAA Chinese Cavendish 5[6 2/10 AAAA IC2 8/10 1/6 AAAA 2390-2 3[4 0/4 AAAA TU8 5/6 0/4 AAAA Calypso 4/4 NT AAB Mysore 8/8 0/5 AAB Lady Finger 5/10 0/10 AAB Sugar 8/8 2/6 AAB Home Plantain 6[6 1 [8 AB Ney Poovan 3/6 0/4 ABB Bluggoe 3/8 0/7 ABB Ducases 4/4 2/6

TOTAL 131/155 19/127

* Survival= number of shoot pieces that survived and produce shoot initials/number of pieces inoculated NT, Not Tested

Results

The effect o f apical domes on survival and growth o f explants

Survival of explants. The rate of survival varied amongst cultivars (Table 1). Of one hundred and fifty five (155) shoot-tip explants with apical domes cultured on IM (85%) survived. In contrast, only 19 (15%) out of 127 survived, where apical domes were removed.

Appearance and rate of growth. Irrespective of the presence or absence of apical domes the colour of survived explants changed from creamy white to green within 7 to 14 days. Shoot initials developed after 30 to 56 days with the apical shoots developing earlier. Cavendish types (genome AAA) formed shoot initials first whereas cv. Lady Finger (genome AAB), cv. Ney Poovan (genome AB) and cv. Bluggoe (genome ABB) were slow to respond and some failed to grow. The reason for this is not known.

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Figure i. Stages of development of banana tissue "cultures derived from shoot-tip explants with intact apices, a) Single shoot formed by elongation of main shoot apex of shoot-tip explant, b) Outgrowth of four additional shoot.:initials from bases of leaf axils (arrows) of shoot-tip explant, c) Cluster of shoots developed from the base of excised shoot in cv. Williams. d) Small but numerous buds formed from the base of excised shoot in cv. Bluggoe.

Type o f growth. Two types of shoots were initiated from explants with intact apical domes. These were: single apical shoots formed from the main apex and the basal rhizomatous tissue swelled (Figure la) and occasionally, outgrowths o f two to four shoots (Figure lb) from the adventitious buds at the bases of the leaf axils [11]. When the apical domes were removed from the explants, two to four shoots developed only from the leaf axils.

The effect o f cytokinin on shoot proliferation and root growth

Shoot proliferation. Shoots did not proliferate if cytokinin was omitted from the medium (Table 2). The cytokinin BA was found consistently more

163

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Figure 2. Effect of BA(a) and kinetin(b) on the root number of seven selected banana cultivars, l~1 , cv. Lacatan; I"] , cv. Lady Finger; ~ , IC2; [~, cv. Ney Poovan;

, cv. Suerier; ~] , cv. Williams; II , cv. Bluggoe. Assessments were made 6 weeks after growth on basal medium containing four levels of cytokinins. The bars represent the SD of the mean obtained from 4 replicates of 10 shoots and repeated in three separate experiments.

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effective than Kn for s h o o t prol i ferat ion even though the extent o f the

response was variable with cultivars. Shoot mul t ip l ica t ion was reduced in most cultivars at the higher cy tok in in levels (10 to 15 m g l -1 ) , except for cv.

Mysore which prol iferated best at 15 rag.1 -x BA (Table 2). Cultivar Williams formed minu te clusters (Hate 3) o f more than 20 well-formed shoots which measured from two to twelve m m in height on all media conta in ing BA. On the other hand , cv. Bluggoe formed compact , nodular masses of tissue

(Figure 1 d) with shootlets rarely exceeding three m m in height on the same

BA media. New shoots arose from the base of excised shoots. Proliferation of shoots

only occurred when rhizomatous base material was included each shoot.

165

in

Root growth. Shoots failed to produce roots or produced stunted, un- satisfactory roots on BM without BA or Kn.

Shoots produced roots within 15 to 20 days when cytokinin was present (Figures 2 and 3). BA at high concentrations (10-15 mg.1-1)was inhibitory and induced severe leaf necrosis three weeks after treatment. The numbers of roots and root lengths were increased when BA was replaced by Kn, but the responses varied amongst cultivars. Cultivars Williams and Bluggoe produced roots only on Kn media. Leaf necrosis was never observed on Kn media.

Transplanting

Rooted plantlets formed on Kn medium were transferred to a peat-sand mixture medium and held in the glasshouse. All plantlets survived and grew normally attaining average height of 30 cm in ten weeks.

Discussion

Previous reports [7, 13] on the importance of apical dominance in shoot production from explants of banana are conflicting. Ma & Shii [7] reported that the destruction of apical dominance by removing the apical domes, was essential for the production of multiple shoot-initials in cv. Cavendish. Swamy et al. [13] later reported that multiple shoot-initials were produced in the presence of apical domes in cv. Robusta. The findings reported here for a range of cultivars support those of Swamy et al. [13] in that the removal of the apical dome is not essential for multiple short initiation. Shoot-tip explants with intact apical domes formed both single apical and multiple lateral shoot-initials. However, explants wihtout apical domes formed only multiple lateral shoots. With or without an apical dome, BA stimulated multiple shoot development, as has been shown for other plants in a comprehensive review by Phillips [9].

The finding that banana explants with apical domes have a higher rate of survival has not been observed before, though this has been reported in other crops [5]. Explants without apical domes offer no particular advantage. The retention of apical domes in explants is therefore recommended for banana tissue culture.

It was found in this study with banana as has been found in other crops [5] that BA was an effective cytokinin for stimulation of shoot proliferation. The rate of shoot proliferation varied amongst different cultivars, confirming an earlier report by Cronauer et al. [2]. It is recommended that BA be used as cytokinin in the multiplciation medium.

A poor proliferation rate was observed with Kn on all cultivars reported

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here but Kn stimulated shoot elongation (unpublished) and induced

vigorous root growth. An intermediate shoot elongation stage is not necessary in banana tissue culture. No supplementary auxin is required for root growth.

The technique reported here has been applied to more than 50 different banana cultivars and six seed-fertile Musa species in this laboratory, thus demonstrating that it is a general method for banana propagation. To date, about five thousand banana plants derived from shoot-tip culture have been produced and grown on in the glasshouse. Some of these have been trans- ferred to disease nurseries in the field for selection o f resistance to Panama disease, (F. oxysporum f. sp. cubense), whilst others have been planted in commerial fields to observe variability. Results of these experiments will be published in due course.

Acknowledgements

I would like to thank Mr. P.E. Mayers, Mr. J. Daniells and Dr Jeff Johns for banana suckers used in this study, and Drs R.C. Colbran, G.M. Behncken and Mr K.G. Pegg for their interest and constant encouragement in this work. This work was supported by the Banana Industry Protection Board of Queensland.

References

1. Bower JP, Fraser C (1982) Shoot tip culture of Williams bananas. Subtropica 3:13-16

2. Cronauer SS, Krikorian AD (1984) Rapid multiplication of bananas and plantains by in vitro shoot tip culture. Herr Science 19:234-235

3. De Guzman EV, Decena AC, Ubalde EM (1980) Plantlet regeneration from unirradiated and irradiated banana shoot tip tissues cultured in vitro. Phil Agri 63: 140-146

4. Hwang SC, Chen CL Lin JC, Lin HL (1984) Cultivation of banana using plantiets from meristem culture. Hor~ Science 19:231-233

5. Hu CY, Wang PJ (1983) Meristem, shoot tip and bud cultures. In: Evans DA, Sharp WR, Ammirato PV, Yamada Y (eds) Handbook of plant cell culture. Volume 1. Techniques for propagation and breeding. MacMillan Publishing Co: New York and London, pp 177-227

6. Jarret RL, Rodriguez W, Fernandez R (1985) Evaluation, tissue culture propagation, and dissemination of 'SABA' and 'PELIPITA' plantains in Costa Rica. Sci Herr 25:137-147

7. Ma SS, Shii CT (1972) In vitro formation of adventitious buds in banana shoot apex following decapitation. J Herr See China 18:135-142

8. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473-497

9. Phillips IDJ (1975) Apical dominance. Ann Rev Plant Physiol 26:341-367 10. Simmonds NW (1966) Banana, 2nd edition. Longmans: London, 512 pp 11. Skutch AF (1932) Anatomy of the axis of the banana. Bet Gaz 93:233-258 12. Stover RH (1972) Banana, plantain and abaca diseases. Commw Mycol Inst, Kew,

UK, 316 pp 13. Swamy RD, Rao NKS, Chacko EK (1983) Tissue-culture propagation of banana.

Sei Herr 18:247-252 14. Vessey JC, Rivera JA (1981) Meristem culture of bananas. Turrialba 31:162-163