6. Biotechnology and in vitro mutagenesis for banana improvement - Mak Chai^[5],Y.W. Ho^[6], K.W. Liew^[7], J. M. Asif^[8]

Abstract

Banana is the second most commonly grown fruit crop in Malaysia. Overall banana production has decreased due to the increasing threat of Fusarium wilt disease, high labour costs and marketing issues. This program was initiated to improve banana cultivars by induced mutations and biotechnology, especially to produce mutant varieties with improved traits such as Fusarium tolerance, short stature plants, early fruiting and high bunch weight. Banana shoot-tip cultures were most suitable for micropropagation for large-scale plant production. Commercial companies have adopted this method, and they produce 1.3 million plants annually, with approximately 0.5% somaclonal variation. However, the cost of production of in vitro plants could be reduced by low-cost micropropagation. Somaclonal variation has been effective in banana for the selection of useful somaclones, e.g. early flowering and tolerance to Fusarium wilt. In Novaria, an early flowering mutant, 7% of the plants survived in the Fusarium 'hot spot' for 3 years. Consequently, somaclonal variation is being used as a strategy to select useful mutants. Since bananas are mostly sterile polyploids, highly heterozygous, and propagated vegetatively, genetic improvement by cross-breeding is an insurmountable task. On the other hand, mutation techniques are highly suitable for banana improvement. Pisang Berrangan (AAA), a popular dessert banana variety, was gamma-irradiated at several dosages (20-60 Gy). The highest percentage of variants, such as changes in leaf coloration and leaf texture, leaf deformation, stunted growth etc., was obtained with 45 Gy treatment. The double-tray system was developed for the selection of mutants tolerant to Fusarium wilt. Among selected plants showing tolerance to Fusarium wilt, none survived field evaluation under Fusarium 'hot spot' conditions. Molecular characterization with RAPD of resistant and susceptible banana types showed random variation for different markers. However, four primers showed bands specific to either resistant or susceptible seed progenies, but could not provide information on the degree of co-dominance.

1. INTRODUCTION

Bananas are grown in 122 countries, with a cultivated area of 3.8 million hectares and a total production of 56.4 million metric tonnes. In Malaysia, banana is the second most widely cultivated fruit, covering about 26,000 ha with a total production of 530,000 metric tonnes. About 50% of the banana growing land is cultivated with Pisang Berangan and the Cavendish type, and the remaining popular cultivars are Pisang Mas, Pisang Rastali, Pisang Raja, Pisang Awak, Pisang Abu, Pisang Nangka and Pisang Tanduk. Bananas are cultivated for local consumption by smallholders, and only about 12% of the total production is exported, mainly to Singapore, Brunei, Hong Kong and the Middle East. However, banana production in Malaysia has decreased because of an increasing threat of diseases (particularly Fusarium wilt), high labour costs and marketing issues.

The present research program was initiated with the objective of improving the important dessert bananas in Malaysia. This includes production of resistance or tolerance to Fusarium wilt or Panama disease, short plant stature, early fruiting, and high bunch weight. Banana cultivars are vegetatively propagated clones and are generally triploids and sterile. Tissue culture techniques have been exploited for (a) propagation of selected lines or natural variants; (b) generation of somaclones; (c) production of meristem pieces for in vitro mutagenesis and polyploidy induction; and (d) zygotic embryo culture to generate seed progenies for genetic and molecular studies.

2. MICROPROPAGATION OF BANANAS

Ma and Shi [1,2] reported in vitro culture of banana. Shoot-tip culture in combination with heat therapy was successfully used to produce virus-free banana plants [3] as well as for mutation induction [4-9]. Several reviews have been written on the in vitro culture techniques for different applications [10-17].

The shoot-tip meristem culture technique is used routinely for large-scale propagation of bananas in Malaysia [18]. Currently, commercial production of micropropagated banana plants in Malaysia is estimated at around 1.3 million. The commercial micropropagation protocol has been successfully modified to bring somaclonal variation down to 0.5% [19]. There are several advantages of tissue culture-derived banana plants, such as vigorous growth, high survival rate, uniformity, and high flowering rate (90% of plants flower compared with 59% of plants produced from suckers). This shortens the harvesting period by about 2 months. Reduction in the cost of micropropagation has been achieved by using locally made culture containers, and by replacing tissue-culture grade sucrose with commercial cane sugar, and Gelrite with locally available agar. We also found that the multiplication rate could be enhanced by 33% by using liquid or semi-liquid media instead of solid medium. We could also achieve rooting by placing plantlets in a hydroponic system in the greenhouse; the resulting rooted plantlets needed a shorter period of hardening. Other cost reduction strategies [20, 21], particularly the use of sunlight, should be pursued to reduce the current production cost further.

3. EXPLOITATION OF SOMACLONAL VARIATION

The occurrence of off-type plants, commonly known as somaclonal variants [22], is relatively common in in vitro plants [12,17,23-30]. Usually, most of the somaclones are undesirable from agronomic point of view. Nevertheless, the phenomenon is useful in generating genetic variation for banana improvement:

(a) With continuous selection of somaclones for earliness from tissue culture-derived commercial plantings of Pisang Berangan, the proportion of plants showing earliness in flowering increased gradually from 16% to over 60% at 8 months of planting (Table 1). This means that at about 11 months after planting, 66% plants were ready for harvest compared with 4% for sucker material and 16% for unselected tissue-cultured plants.

Table 1 Selection of somaclones for early flowering in Pisang Berangan

(b) Using tissue-culture-derived plants, 27% of Pisang Rastali (one clone) survived for more than 3 years in the Fusarium wilt-infested field, while all other clones planted at the same time had succumbed to the disease. Dissection showed the symptoms of Fusarium infection at the base of the corm tissue. The vascular discoloration was arrested 20-30 cm above the base. The continuous selection of tolerant plants has resulted in the release of a Fusarium wilt-tolerant clone of Pisang Rastali called Mutiara [18]:

Table 2 Survival of Pisang Rastali in a Fusarium 'hot-spot'

Table 3 Screening somaclonal variants in the Fusarium 'hot spot' for disease tolerance

Novaria, an early flowering mutant of 'Grande Naine' [7], was one of the susceptible controls used. It was encouraging to note that 7% of the plants survived in the Fusarium 'hot spot' for 3 years. Consequently, exploitation of somaclonal variation has been adopted as a regular strategy to select for Fusarium-wilt resistance in Novaria and Pisang Berangan (Table 3). It was also shown that lines derived from different suckers of the same mother plant tended to show variation in degree of tolerance (Table 4).

Table 4 Line variation in response to Fusarium wilt tolerance in the 'hot spot'

4. IN VITRO MUTATION BREEDING

Edible bananas are mostly sterile polyploids and must be propagated vegetatively, hence genetic improvement through cross-breeding is not possible. Mutation breeding has been suggested as an excellent alternative approach for banana improvement [4,5,7-9,16,31-40]. In addition, the heterozygosity of asexual banana clones makes them suitable for mutation induction. The heterozygotic status is expected to be Aa in loci of diploid cultivars while the triploids of A genomic types can exist in either Aaa or Aaa forms. For interspecific hybrids, the heterozygotic constitution could be AaB, Aab, AAb, ABb, aBb or aBB. Mutation induction may uncover a recessive phenotype by mutating, inhibiting or deleting the corresponding dominant allele [25].

The use of cultured shoot tips for mutagenesis has facilitated mutation induction and the regeneration of potential mutants [8,26,41]. An early flowering mutant of Grand Naine, GN-60Gy A, which also showed differences in the zymograms of soluble proteins and esterase isozymes, was induced by Novak [41] after exposing shoot tips to gamma radiation. Further selection of GN-60Gy A in Malaysia has resulted in the release of an early fruiting Cavendish banana called Novaria [7]. Matsumoto and Yamaguchi [42] selected an aluminium-tolerant mutant from irradiated protocorm of a Cavendish banana.

Pisang Berangan (AAA) is a popular dessert banana, having good fruit quality, flavour, colour, pulp texture, size and shelf life. However, it is relatively tall and very susceptible to Fusarium wilt (Fusarium oxysporium f.sp cubense) and freckle disease caused by Cladosporium musae. Consequently, a mutation breeding program was initiated using gamma irradiation to induce genetic variation so that plants could be selected with one or more of the following characteristics: (a) tolerance to Panama disease; (b) short plant stature; and (c) early fruiting and high bunch weight. The present review aims to present the current status of the research, problems encountered, and research strategies.

4.1. Techniques for in vitro mutation induction

Healthy and normal plants from selected Pisang Berangan were micropropagated to produce shoots/buds with apical domes from which meristem pieces (about 1 cm × 2 mm) were aseptically excised. About 20 to 30 meristem pieces were transferred to each sterile Petri dish, which contained a piece of sterile moist paper, and then sealed with parafilm. The shoot-tip meristems in the Petri dishes were irradiated in a gamma cell with a ⁶⁰Co source to give the required doses. The treated explants were washed thoroughly with sterile distilled water prior to culture on modified MS medium [43] supplemented with 150 ml/l coconut water, 30 g/l sucrose, 5 mg/l BA and 5 g/l agar (A02 Type 900). Subsequently, each growing shoot was separately cultured to generation M₁V₄ (three subcultures at monthly interval) and then allowed to root (3 to 4 weeks). As mutation is a single cell event, mutation induction in a multi-cellular shoot-tip would result generally a chimera in the M₁ plants. Hence, mutated cells may be recovered through repeated in vitro propagation of mutated cell progenies in several vegetative cycles. The rooted plantlets were transplanted to soil mix in perforated polythene bags [5" × 7" (approx. 12.5 × 18 cm)] for acclimatization (8-10 weeks) before field planting at 2.4 × 24 m. Evaluation of mutagenic effects was conducted on both nursery and field-grown plants.

4.2. Dose sensitivity and mutagenic effects

A pilot experiment was conducted using doses of 0, 25, 35,45 and 60 Gy of gamma irradiation to study radiation sensitivity of Pisang Berangan [8]. Both survival and shoot multiplication rate decreased with an increase of radiation dose (Table 5). The LD₅₀ dose (the radiation dose that reduces the growth to 50% of that of the control) for Pisang Berangan was 38 Gy. This is in agreement with Novak [41], who found LD₅₀ doses of 25 Gy (2n plants), 35 Gy (3n plants, AAA), 40 Gy (AAB and ABB plants), and 50 Gy (4n plants, AAAA). Whether LD₅₀ dose is a useful criterion for mutation induction is debatable. According to the available information [44], the doses being used currently range from 20 to 60 Gy of gamma irradiation. The general recommendation is to use a low dose on a very large number of primary explants (e.g. 10,000) followed by one subculture in vitro, or a high dose on a large number of primary explants (e.g. 2000) followed by at least three subcultures to M₁V₄. The present radiosensitivity results indicated that the highest percentage of variants was obtained at 45 Gy, but that at 60 Gy many lethal or abnormal mutants would probably not have survived.

Table 5 Survival and multiplication rate per explant at 12 weeks after gamma irradiation

^a No. of buds/shoots produced/explant

Mutagenic treatments caused many morphological changes in both nursery and field-grown plants. These phenotypic variants were increased 4- to 6-fold by gamma irradiation (Table 6). Some of these variants include:

(a) Changes in leaf coloration which included discoloration, darker green, and various forms of leaf variegation;

(b) Leaf deformation, such as uneven lamina; leaf becoming smaller, narrower, long but weak, broad and oval; tendency to roll up at edges;

(c) Changes in leaf texture such as waxy, leathery or rough, crumpling and rough veins;

(d) Pseudostem becoming flat or stiff or bulbous;

(e) Various degrees of stunted growth.

Table 6 Variability induced by gamma irradiation of Pisang Berangan

^a % of plants harvested at 8 weeks earlier than the mean
^b % of plants with 250 cm high or less

Additional phenotypic variants observed in field-grown plants include:

(a) Stunted growth or choking appearance;

(b) More compact and erect leaves;

(c) Pseudostem becoming flat, stiff or even split and its colour varying from dark brownish (more anthocyanin) to lighter yellow with waxy petiole;

(d) Various forms of deformation in bunch and fruit characteristics.

For the two important agronomic traits, namely number of weeks to harvest and plant height at flowering, the mean values of irradiated plants did not differ significantly from the control plants. However, mutation induction tended not only to increase the variability, as indicated by the increase in the coefficient of variation (CV), but also showed a much higher frequency for plants having early fruiting or shorter height (Table 6). The amount of variability observed for these two quantitative traits may be underestimated, because many mutagenised plants might not survive to maturity due to the adverse effects of mutagenic treatments.

The results have demonstrated that mutation breeding coupled with in vitro technique could produce morphological changes as well as an increase of variability in quantitative traits. Though many morphological changes are undesirable, this source of induced variation is potentially useful for the sterile Musa species.

4.3. Screening for desirable traits

4.3.1. Screening for agronomic traits

As mutation is a random and rare event and mutants are generally recessive and deleterious, mutation breeding requires the screening of a large sample of mutagen-treated populations to identify the desired individuals. Screening has to be considered at the different stages of growth development. Field screening of large populations, particularly for agronomic traits, was impractical in view of the space required, the management needed, and the 2-year cycle needed for selection. Phenotypic selection of M₁ plants for important agronomic traits, such as early fruiting, shorter plant stature and high bunch weight, was unreliable because the minor variations among plants are confounded with the deleterious effects of mutagens and with environmental factors such as soil fertility, water stress and management practices.

4.3.2. Screening for disease tolerance in mutated materials

Panama disease caused by the soil-borne fungus Fusarium oxysporium f.sp. cubense is an important factor limiting commercial cultivation of bananas in Malaysia. Control measures involving field sanitation, soil fumigation and liming are not effective. Although the fungal infection starts with invasion of the roots before ingress into the vascular system, the expression of visible symptoms results from occlusion by the fungus mycelium, induction of tyloses, enzymatic breakdown of parenchymatous tissues and the action of fusaric acid, a potent mycotoxin, produced by the fungus. Mechanisms of resistance are believed to be multi-faceted, and plant age and vigour are contributing factors.

To facilitate disease screening, a quarantine field heavily infested with the pathogen was established as a Fusarium 'hot spot' in an oil palm plantation. This facility allowed complete disease screening at nursery and field planting stages. Eight- to ten-week-old nursery plants derived from gamma-irradiated materials were planted in the Fusarium 'hot spot' to screen for disease resistance. In the early pilot experiment to study radiation sensitivity, 2000 plants were planted in a field which was later heavily infected with Fusarium wilt. All plants eventually died except one, which survived for 2 years in the field. This plant, derived from 45 Gy gamma-ray treatment, was designated as I289. Two suckers from this selection were micropropagated to produce plants for further confirmatory testing against the Fusarium pathogen. However, the results indicated that the tolerance of this selection had broken down, as the survival rates were only slightly higher at different stages of assessment those of the control (Table 7). This may indicate that I289 was merely an 'escape', or that the pathogen population in the 'hot-spot' is too high to allow the survival of any tolerant plants.

Table 7 The survival of plants derived from I289 in the Fusarium 'hot spot'

As tolerance to Fusarium wilt appears to show quantitative variation, an attempt was made to irradiate in vitro meristems derived from I289 at 40 Gy with the hope of increasing the degree of tolerance by accumulating mutagenic changes of minor genes for Fusarium wilt tolerance. A total of 49 plantlets were produced and planted in the Fusarium 'hot spot' in 1996. A few plants showed mild leaf yellowing after 2 months, but all succumbed to the disease eventually.

In another experiment, 4000 plants derived from 35 Gy gamma irradiation were planted in a disease-free area; 1818 suckers were taken from normal fruit-bearing plants and tested in the Fusarium 'hot spot'. Five plants were found to survive after one year, but then succumbed to the disease.

Results of screening other batches of gamma-irradiated plants in the Fusarium 'hot spot' are shown in Table 8. From all the screening trials, it was generally observed that plants of 4-5 months old became very susceptible. Susceptible plants generally did not survive for more than 10 months in the field.

Table 8 The survival of gamma-irradiated Pisang Berangan in the Fusarium 'hot spot'

After screening about 12,000 mutagenic plants, no resistant plants had been identified. Though some selections appeared promising initially, they became susceptible under prolonged testing. It appears that the inoculum concentration in the Fusarium 'hot spot' may be too high to allow the survival of moderately resistant plants. As tolerance to Fusarium wilt appears to show quantitative variation, the 'hot spot' may not be effective in the identification of tolerant lines controlled by minor genes.

4.3.3. Development of techniques for screening resistance to Fusarium wilt disease - the 'double-tray System'

Field evaluation is the most reliable method for disease resistance screening, but is demanding in terms of cost, manpower and space requirements [45]. There is also the need to maintain strict quarantine control to avoid pathogen spread. In addition, plants tend to show symptoms only after 4-5 months [46]. The uneven distribution of pathogen in the field can lead to 'disease escape', while many variables that can affect infection and symptom expression cannot be altered or controlled. After two years of experimental trial, an alternative method of screening seedlings at the nursery stage has been developed. [47]. The technique is a modification of the double-cup sand-culture containment method developed earlier for laboratory testing of pathogen virulence [48]. The double cup was replaced by a 'double-tray system'.

The double compartment consists of a tray measuring 43 × 29 × 9 cm that fits snugly into another, larger, outer tray measuring 46 × 31 × 20 cm (Plate 2). The upper tray was three-quarters filled with sterilized fine river sand. The plantlets were watered with tap water every other day plus Hougland's solution once a week. After about 45 days of acclimatization, the test plantlets were carefully uprooted and only those with healthy white roots were selected for inoculation by immersion in the appropriate conidia suspension for 2 h before being tagged and replanted in the trays for maintenance and observation in the greenhouse.

Table 9 Response of some banana cultivars to Fusarium oxysporum f. sp. cubense (FOC)

R, resistant; T, tolerant; S, susceptible; VS, very susceptible.

Two-month-old plantlets (10-15 cm height) were found to be suitable for differential expression of symptoms, similarly to field evaluations at the Fusarium 'hot spot'. Seedlings with heights of less than 10 cm did not produce consistent symptoms.

Race 4 of Fusarium oxysporum f. sp. cubense was freshly isolated from susceptible 'Novaria' plants on Potato Dextrose Agar [49] followed by single spore isolation [50] and inoculum preparation in Armstrong's Liquid Medium [49]. Cultures were incubated at room temperature and shaken twice a day for 7 days, after which they were filtered through two layers of cheesecloth. Inocula at the desired concentrations, comprising mostly microconidia, were then prepared with the aid of a haemocytometer, and immediately used for plantlet-root inoculation. Root immersion for 2 h in the pathogen suspension was found to be effective. All susceptible plants produced foliage and rhizome symptoms at inoculum concentrations higher than 5 × 10² spores/ml. All susceptible plants tested, namely Pisang Berangan and Novaria, showed both foliage and rhizome symptoms within a period of 10-28 days. However, more than 94% of tolerant plants (namely Goldfinger and Mutiara), as well as all the uninoculated control plants, did not show any symptoms.

This 'double-tray' technique is being used for preliminary screening of somaclonal variants and induced mutants. Meanwhile, the response of some banana cultivars to Fusarium oxysporum f. sp. cubense was also noted (Table 9).

4.3.4. Induction and performance tetraploid Pisang Mas

In Malaysia, Pisang Mas, which belongs to AA group, is well known as one of the most popular local dessert cultivars. Its synonyms are 'Amas' (Philippines) and 'Kluai Khai' (Thailand). The dessert banana has potential for local and export markets. However, it has a low yield (an average of 8-12 kg/bunch), its skin is thin, and its fruit size is small. Therefore, the potential of polyploidy for the improvement of Pisang Mas was studied [51]. The induced tetraploid might also be used for crossing to a selected diploid to produce a secondary triploid.

Shoot meristem pieces obtained from in vitro cultured plantlets were immersed in shallow 0 (control), 0.25, 0.5. 0.75, and 1.0 % (w/v) aqueous colchicine solutions for 1, 2, 3 or 4 hours by placing on an orbital shaker (100 r.p.m.) at room temperature (25-28ºC). Sterile distilled water was used as a control instead of colchicine. After treatment the meristem pieces were washed with sterile distilled water and cultured on semi-solid modified Murashige and Skoog (MS) medium [43] supplemented with 30 g/l sucrose, 0.05 g/l citric acid and 2 mg/l BA, at pH 5.7. Each meristem piece was propagated every 3-4 weeks for three vegetative cycles. Ploidy levels were then determined by flow cytometry, and the tetraploid plants were confirmed by chromosome counts. A high level of tetraploids was induced with 1.0% colchicine treatment for 3 h (41.18%). However, this treatment caused heavy damage and the casualty rate was 27.03%. The treatment that gave high tetraploidy (15%) with low mortality (7.48%) was 0.5% colchicine for 2 h. For all treatment combinations, the rate of tetraploid induction varied from 2.6% to 37.5% and mixoploidy ranged from 14% to 47.6%.

The histograms of different ploidy level of the plants are shown in Figure 1. The peak at the left represents debris. Nuclei released from a diploid plantlet appeared at channel 50 for G₁ phase (Figure 1a). Plantlets with double the number of chromosomes have their G₁ peak at channel 100 (Figure 1b), and are therefore tetraploid. Too high a concentration or too long a duration of colchicine treatment might result in higher ploidy levels. Figure 1c shows the histogram of octoploid nuclei, which peak at channel 200. Mixoploid plants consisting of both diploid and tetraploid, or tetraploid and octoploid cells were also observed (Figure 1d & e).

Chimeric plants were produced by even shorter time treatments with low concentrations of colchicine. By prolonging the treatment, more cells in a single meristem can be exposed for chromosome doubling. However, overtreatment may result in a ploidy level higher than tetraploid and increased mortality. Colchicine has direct toxic effects and acts as a strong killing agent even at millimolar concentrations. The chimerism would probably be inherited or gradually revert to diploidy through continuous micropropagation.

Figure 1 Histograms of Pisang Mas treated with colchicine. (a) control; (b) tetraploid; (c) octoploid; (d) mixoploid 2n + 4n; and (e) mixoploid 4n + 8n.

The length of the stomata in diploids is about twice that in tetraploids. However, mixoploid plants are intermediate between diploid and tetraploid plants. Breadth measurements show that 22.0% of mixoploids are diploid while 77.0% are tetraploid. However, length measurements show that 4.0% of the mixoploids are diploid and 74.5% are tetraploid. The adaxial leaf surface has significantly fewer stomata than the abaxial. The stomata densities of mixoploids and tetraploids were significantly different from the control (P < 0.05). However, the stomata densities on both the adaxial and abaxial surfaces of the diploids and mixoploids overlapped.

The octoploid derived from the doubling of the tetraploid tends to show stunted growth. The octoploid had a big pseudostem and grew very slowly compared with the diploid. The multiplication rates for diploids and tetraploids were 1.02 ± 0.33 and 0.89 ± 0.38 respectively. Slower growth rates and delayed appearance of buds or shoots were also observed in other polyploid studies [52, 53]. After 4 weeks, tetraploid plants have a big pseudostem, shorter stature with a thicker leaf, and are a slightly darker green than diploid plants. Comparisons between diploid and tetraploid plants are shown in Table 10. All parameters measured were significantly different between diploid and tetraploid plants (P < 0.05) except the primer root diameter.

Table 10 Morphology of diploid and tetraploid plants after 4 weeks in culture

ns, non significant at P < 0.05
^a significant at P < 0.05

In the field, the tetraploid plants showed drooping leaves and non-compact pseudostems with sucker production. However, the plants tended to be more vigorous and taller, with bigger girth, but smaller bunch size than diploids (Table 11).

Table 11 Field performance of tetraploid Pisang Mas

^a 50% of the plants remained non-fruiting after 15 months in the field.

4.4. Studies on wild banana, Musa acuminata ssp. malaccensis

Malaysia is one of the centres of diversity for both cultivated and wild bananas. Co-evolution of host and pathogen is believed to give rise to the diversity and complexity of Fusarium wilt disease. Musa acuminata ssp malaccensis (AA) shows good bunch and fruit characteristics. It is highly fertile and the fruit is gummy with plenty of seeds. Research has been undertaken to use seed progenies for variability characterization based on flow cytometric analysis of ploidy and nuclear DNA content [54] and anthocyanins [51], polyploidy induction [54] and genetic studies on Fusarium wilt resistance.

4.4.1. In vitro zygotic embryo culture

Due to low seed germination under natural soil conditions (2% in a greenhouse study), in vitro zygotic embryo culture was developed [51]. Seeds were soaked in 1.4% (v/v) sodium hypochlorite solution for 10 min followed by a quick wash with 70% (v/v) ethanol. Embryos were removed and cultured in glass jars (60 × 80 cm), each jar containing ten embryos. The culture medium consisted of MS salts supplemented with nicotinic acid (0.125 mg/l), ascorbic acid (0.2 mg/l), thiamine HCl (0.5%/l), pyridoxine HCl (0.125 mg/l), myoinositol (2.5 mg/l), glutamine (150 mg/l) and sucrose (5%).

Table 12 Factors affecting the growth of in vitro grown embryos

^a treatment means are significantly different

In vitro embryo culture increased the germination rate to more than 90%. However, there were seeds in which either endosperm or embryos or both were absent. Vuylsteke and Swennen [55] explained that the low germination of mature triploid Musa seeds was caused by embryo malformation. The effects of lighting conditions, gelling agent and embryo orientation on some growth conditions were observed for a period of one month (Table 12). Darkness delayed shoot emergence (8.9 days) as compared to light (7.3 days). Dark-grown embryos produced longer shoots (3.9 cm) and roots (5.0 cm) than light-grown embryos. Similarly embryos grown in the dark produced more roots (11.0) than light-grown embryos (7.0). Raghavan and Torrey [56] reported that 12 h light and dark cycles inhibited root growth. However supplementing the medium with low concentrations of IAA promptly overcomes this inhibition. This may imply that light causes the inactivation of an auxin-like substance necessary for root formation in embryos

Gelling agent had no significant effect on shoot length or number of roots per seedling. Embryos grown on Gelrite produced shoots earlier (5.5 days) compared to agar (7.6 days). A similar trend was observed for root emergence where roots appeared in 6 days on Gelrite compared to 9 days on agar. Roots were significantly longer on Gelrite (10.5 cm) than agar (6.6 cm). These might be related to the presence of impurities in the agar [57, 58].

Embryo orientation did not have a significant effect on the number of days to shoot emergence, number of roots, or root length. However, long shoots (5.5 cm) were produced by completely exposed embryos. Roots developed much earlier (7 days) with fully exposed embryos, followed by embryos partially embedded (8-10 days) whereas embryos embedded fully in the medium required more time (14 days) to develop roots. The best morphological characteristics were obtained when the haustorium end touched or was slightly embedded in the media. Afele and De Langhe [59] reported that embryos of Musa balbisiana with their longitudinal axis laid flat and half-embedded on solidified agar medium produced the highest germination and the most desirable plantlet characteristics. They obtained a germination rate of 94% in vitro within 7 days, but only 50% germination after 54 days for greenhouse-grown seeds.

4.4.2. Genetic studies on Fusarium tolerance

Ten to 15 suckers of Musa acuminata ssp. malaccensis were collected from five locations and planted in the Fusarium 'hot spot' with Pisang Berangan randomly planted among the tested suckers as a susceptible control. Results showed that all Pisang Berangan plants succumbed to Fusarium wilt disease within 4-5 months, whereas all suckers survived for more than 2 years without any disease symptoms. Dissection of the rhizomes also revealed no disease symptoms (Table 13).

Table 13 Response of the wild Musa acuminata ssp. malaccensis suckers to FOC race 4 after 2 years in the Fusarium 'hot spot'

Seed progenies from the above five populations were produced through in vitro zygotic embryo culture and each individual was micropropagated to produce clones for screening Fusarium wilt tolerance using the 'double-tray' system. Seedlings from different populations showed variable response to FOC race 4. Within each progeny, segregation was observed for Fusarium wilt tolerance (Table 14). Visible symptoms appeared 2-3 weeks after inoculation. Leaf chlorosis started with the older leaves and then progressed upward to the younger ones. Occasionally, a split pseudostem was seen. Plants that showed severe foliar yellowing also had extensive rhizome discoloration. The highly susceptible controls died within 4 weeks.

Table 14 Seed progenies showing segregation for Fusarium wilt tolerance

ns, not significant at 5%

The different genetic ratios observed among five populations suggested that the material used was very diverse genetically. The five populations were obtained from different areas and originated from different stocks of mother plants. Hence they may be different in their genomic compositions and thus their response to FOC. It appears that the seed progenies were very heterogeneous, as wild bananas are both out- and inbreeders. Wild bananas are thought to have coevolved with the FOC pathogen, and their differential response might also be a reflection of the complexity of the pathogen, as indicated by the diversity of the VCG complexes isolated in Malaysia [45, 60].

4.4.3. RAPD marker characterization of resistant and susceptible seedlings

Leaf samples were collected and DNA extracted from five resistant (R) and five susceptible (S) clonal seedlings of Musa acuminata ssp. malaccensis. Initially 28 arbitrary primers, 10 bases in length, were evaluated, of which only 15 produced DNA products. Good amplification was obtained with a concentration of 1.5 mM of MgCl₂.

A total of 96 major scorable amplification products (bands) from 15 primers were obtained from the ten resistant (R) and susceptible (S) seedlings. Only 10 out of 96 markers were monomorphic and shared among the seed progenies, whereas the remaining 86 markers were polymorphic. The number of scorable RAPD fingerprints generated per primer varied from 4 to 9, with an average of 6 markers per primer. In general, the amplified DNA fragments ranged from 200 bp to 1.5 kbp.

The variation observed for different markers was randomly present in both resistant and susceptible seed progenies. The markers specific to resistant and susceptible seed progenies were only partially resolved by most of the primers used. However, there were four primers that showed bands specific to either resistant and susceptible seed progenies. Seed progenies amplified with the Primer OPA-03 showed a major band of 500 bp shared by all susceptible plantlets that was absent in resistant progenies (Figure 2a). A band of 1000 bp derived from the Primer 21 was observed to be present only in the resistant seed progenies but absent in the susceptible ones (Figure 2b). In the case of Primer 27, a band of 1.5 kbp was shared by four of the five susceptible plantlets but was absent in all resistant progenies (Figure 2c). For Primer 24, a major band of 400 bp was observed in susceptible progenies but was absent in all resistant plantlets (Figure 2d). The remaining primers showed high polymorphism, but without specificity to either resistant or susceptible plantlets. These results demonstrated that RAPD is a robust technique for characterizing seed progenies, but it did not provide any information about the degree of co-dominance. Hence, they may be of low utility for the study of segregating populations where information about heterozygous individuals is very vital. Furthermore, the repeatability of these RAPD results needs to be established.

Figure 2 Results using RAPD primers, showing bands specific to resistant and susceptible plants of seed progenies. (a) Primer OPA-03 (5'-AGTCAGCCAC-3') - a major band of 500 bp is found only in susceptible (S) plantlets; (b) Primer 21 (5'-CGCTGTCCTT-3') - a major band of 1000 bp is present only in Fusarium wilt-resistant (R) plantlets; (c) RAPD primer 27 (5'-CTCCGCCA-3') - a major band of 1500 bp is present only in Fusarium wilt-susceptible (S) plantlets; and (d) RAPD primer 24 (5'-GTGCGTATGG-3') - a major band of 400 bp is found only in Fusarium wilt-susceptible (S) plantlets.

REFERENCES

[1] MA, S.S., SHII, C.T., In vitro formation of adventitious buds in banana shoot apex following decapitation, J. Chinese Soc. Hort. Sci. 18 (1972) 135-142.

[2] MA, S.S., SHII, C.T., Growing banana plantlet from adventitious buds, J. Chinese Soc. Hort. Sci. 20 (1974) 6-12.

[3] BERG, L.A., BUSTAMANTE, M., Heat treatment and meristem culture for the production of virus free bananas, Phytopathology 64 (1974) 320-322

[4] DE GUZMAN, E.V., et al., "Banana and coconut in vitro cultures for induced mutation studies", Improvement of vegetatively propagated plant and tree crops through induced mutations, IAEA, Vienna (1976) 33-54.

[5] DE GUZMAN, E.V., et al., Plantlet regeneration from unirradiated and irradiated banana shoot tip tissue cultured in vitro, Philippine Agric. 63 (1980) 140-146.

[6] NOVAK, F.J., et al., Mutation induction by gamma irradiation of in vitro cultured shoot tips of banana and plantain (Musa cvs), Trop. Agr. (Trinidad) 67 (1990) 21-28.

[7] MAK, C., et al., Novaria - a new banana mutant, InfoMusa 5(1) (1996) 35-36.

[8] MAK, C., et al., Mutation induction by gamma irradiation in a triploid banana Pisang Berangan, Malaysian J. Sci. 16A (1995) 77-81.

[9] MAK, C., et al., Origin and performance of an early flowering Cavendish banana - Novaria, (Proc. Int. Conference on Tropical Fruits, 23-26 July, Kuala Lumpur) (1996) 119-124.

[10] CRONAUER, S.S., KRIKORIAN, A.D., "Banana (Musa spp)", Trees I. Biotechnology in Agriculture and Forestry. Volume 1 (BAJAJ, Y.P.S., Ed.), Springer Verlag, Berlin (1986) 233-251.

[11] HWANG, S.C., et al., Cultivation of banana using plantlets from meristem culture, HortScience 19 (1984) 231-233.

[12] ISRAELI, Y et al., "In vitro culture of bananas", (GOWEN, S., Ed.), Bananas and Plantains. Chapman & Hall, London (1995) 147-175.

[13] JARRET, R.L., "In vitro propagation and genetic conservation of bananas and plantains", IBPGR Advisory Committee on in vitro storage. Report of the third meeting (Appendix). IBPGR, Rome, Italy. (1996).

[14] KRIKORIAN, A.D., CRONAUER, S.D., "Banana", Handbook of plant cell culture (SHARP, W.R., et al., Eds), Vol.2, Macmillan, New York. (1984) 327-348.

[15] KRIKORIAN, A. D., CRONAUER, S., Aseptic culture technique for banana and plantain improvement, Economic Bot. 38 (1984) 322-331.

[16] NOVAK, F.J., MICKE, A., "Advancement of in vitro mutation breeding technology for bananas and plantain", In vitro mutation breeding of bananas and plantain. First FAO/IAEA Research Co-ordination Meeting on mutation breeding of bananas and plantain, Vienna, Austria (1990) 56-65.

[17] VUYLSTEKE, D.R., Shoot tip culture for the propagation, conservation and exchange of Musa germplasm, IBPGR, Rome (1989) 1-56.

[17a] VUYLSTEKE, D.R., et al., Somaclonal variation in plantains (Musa spp., AAB group) derived from shoot tip culture, Fruits 46 (1991) 429-439.

[18] HO, Y.W., et al., "Micropropagation of planting materials with special reference to banana", Proceedings of Seminar on the Fruit Industry in Malaysia, 7-9 Sept. Johore Bahru (1993).

[18] HWANG, S., KO, W.H., "Somaclonal variation of bananas and screening for resistance to Fusarium wilt", Banana and Plantain Breeding Strategies. (PERSLEY, G.J., DE LANGHE, E.A., Eds), (Proceedings of an international workshop held at Cairns, Australia, 13-17 October 1986), ACIAR Proceedings. No. 21. (1987) 151-156.

[19] HO, Y.W., SINGH, G., "United Plantations Berhad's contribution to the banana industry", Proceedings of the First National Banana Seminar (23-25 Nov. 1998, Awana Genting, Malaysia) (1999) 39-44.

[20] KODYM, A., ZAPATA-ARIAS, F.J., Natural light as an alternative light source for the in vitro culture of banana (Musa acuminata cv.Grande Naine). Plant Cell Tissue Organ Cult. 55 (1999) 141-145.

[21] KODYM, A., ZAPATA-ARIAS, F.J., Low cost alternatives for the micropropagation of banana, Plant Cell Tiss. Organ Culture (2001) (in Press).

[22] LARKIN, P.J., SCOWCROFT, S.C., Somaclonal variation- a novel source of variability from cell culture for plant improvement. Theor. Appl. Genet. 60 (1981) 197-214.

[23] HWANG, S., KO, W.H., "Somaclonal variation of bananas and screening for resistance to Fusarium wilt", Banana and Plantain Breeding Strategies. (PERSLEY, G.J., DE LANGHE, E.A., Eds). Proceedings on an international workshop held at Cairns, Australia, 13-17 October 1986. ACIAR Proceedings. No. 21 (1987) 151-156.

[24] ISRAELI, Y., et al., Qualitative aspects of somaclonal variation in banana propagated by in vitro techniques, Sci. Hort. 48 (1991) 71-88.

[25] NOVAK, F.J., "Musa (Bananas and Plantains)", Biotechnology of perennial fruit crops. (HAMMERSCHLAG, F.A., LITZ, R.E. Eds), CAB International, Wallingford, UK (1992) 449-488.

[26] REUVENI, O., et al., Factors influencing the occurrence of somaclonal variations in micropropagated bananas, Acta Hort. 336 (1992) 367-364.

[27] SMITH, M.K., A review of factors influencing the genetic stability of micropropagated bananas, Fruits 43 (1988) 219-223.

[28] STOVER, R.H., "Somaclonal variation in Grande Naine and Saba bananas in the nursery and field", Banana and Plantain breeding strategies, (PERSLEY G.J., DE LANGHE, E.A., Eds), ACIAR Proceedings No. 21 (1987) 125-136.

[29] VUYLSTEKE, D.R., "Field performance of banana micropropagules and somaclones", Somaclonal variation and induced mutations in crop improvement, (JAIN, S.M., et al., Eds), Kluwer Academic Publishers (1998) 219-231.

[30] JAIN, S.M., Tissue culture-derived variation in crop improvement. Euphytica 118 (2001) 153-166.

[31] DE GUZMAN, E.V., "Project on production of mutant by irradiation of in vitro cultured tissues of coconuts and bananas and their mass propagation by tissue culture techniques", Improvement of vegetatively propagated plants through induced mutation, Technical Document 173, IAEA, Vienna (1975) 53-76.

[32] DE GUZMAN, E.V., et al., "Production of mutants by irradiation of in vitro cultured tissues of coconut and banana and their mass propagation by the tissue culture technique", Induced mutations in vegetatively propagated plants II. IAEA, Vienna (1982) 113-118.

[33] KAO, D.I., Induction of mutations in banana, J. Chinese Soc. Hort. Sci. 25 (1979) 297-306.

[34] MENENDEZ, T., "Application of mutation methods to banana breeding", Induced mutations in vegetatively propagated crops, IAEA, Vienna (1973) 75-83.

[35] MENENDEZ, T., "A note on the effect of ethyl methane sulphonate on Musa acuminata seeds", Induced mutations in vegetatively propagated crops, IAEA, Vienna (1973) 84-90.

[36] NOVAK, F.J. et al., "Potential for banana and plantain improvement through in vitro mutation breeding", ACORBAT Memorieas VIII reunion. CATIE, Turrialba (1987) 67-70.

[37] PANTON, C.A., MENENDEZ, T., "Possibilities and implications of mutation breeding in Jamaica", Induced mutation and plant improvement, IAEA, Vienna (1972) 61-65.

[38] SMITH, M.K., et al., "In vitro mutation breeding for the development of banana with resistance to Race 4, Fusarium wilt (Fusarium oxysporium f.sp. cubense)", In vitro mutation breeding of bananas and plantains, IAEA-TECDOC-800, (1995) 37-44.

[39] SITI HAWA, J.A.H. ZAKRI, et al., "In vitro mutation breeding methodology for the selection of Fusarium wilt resistance in Musa cv. Rastali (AAB)", (Proc. Int. Conference on Tropical Fruits, 23-26 July, Kuala Lumpur) (1996) 125-136.

[40] VELEZ FORTUNO, J., CERDENO MALDONA, A., "The use of radiation in breeding banana (Musa sapientum L)", Induced mutations and plant improvement, IAEA, Vienna. (1972) 485-499.

[41] NOVAK, F.J., et al., Mutation induction by gamma irradiation of in vitro cultured shoot tips of banana and plantain (Musa cvs), Trop. Agr. (Trinidad) 67 (1990) 21-28.

[42] MATSUMOTO, K., YAMAGUCHI, H., Selection of aluminium-tolerant variants from irradiated protocorm-like bodies in banana, Trop. Agr. (Trinidad) 67 (1990) 229-232.

[43] MURASHIGE, T., SKOOG, F., A revised medium for rapid growth and bioassay with tobacco tissue culture, Physiol. Plant. 15 (1962) 473-497.

[44] IAEA, In vitro mutation breeding of bananas and plantains, IAEA-Tecdoc-800, Vienna. (1987) 86.

[45] PEGG, K.G., et al., Fusarium wilt of banana in Australia: a review, Aust. J. Agr. Res. 47 (1996) 637-650.

[46] MORPURGO, R., et al., Selection parameters for resistance to Fusarium oxysporum f. sp. cubense race 1 and race 4 on diploid banana (Musa acuminata Colla), Euphytica 75 (1994) 121-129.

[47] MOHAMED, A.A., et al., "Early evaluation of banana plants at nursery stage for Fusarium wilt tolerance", Banana fusarium wilt management: towards sustainable cultivation. (MOLINA, A.B. et al., Eds), Proc. of the Int. Workshop on the banana Fusarium wilt disease (Genting Highlands Resort, Malaysia, 18-20 October 1999) (2000) 174-185.

[48] LIEW, K.W., "Screening for disease resistance in banana plantlets against Fusarium wilt", Regional Training Course on Molecular approaches, mutation and other biotechnologies for the improvement of vegetatively propagated plants. Universiti Kebangsaan Malaysia (28 Oct.-8 Nov. 1996).

[49] SINGLETON, L.L., et al., "Appendix", Methods for Research on Soilborne Phytopathogenic Fungi (SINGLETON, L.L., et al., Eds), APS Press, St. Paul, Minnesota (1992).

[50] BOOTH, C. Fusarium: Laboratory guide to the identification of the major species, Commonwealth Mycological Institute, Kew, Surrey, England (1977).

[51] AZHAR, M., et al., Polyploidy induction in Pisang Mas, a diploid banana (Musa spp), (Proc. First National Banana Seminar, Awana Genting, Malaysia. 1998) (1999) 134-143.

[52] HAMILL, S.D., et al., In vitro induction of banana autotetraploids by colchicine treatment of micropropagated diploids, Aust. J. Bot. 40 (1992) 887-896.

[53] VAN DUREN et al., Induction and verification autotetraploid in diploid banana (Musa acuminata) by in vitro techniques, Euphytica 88 (1996) 25-34.

[54] ASIF, M.J., et al., Characterization of Malaysian wild bananas based on anthocyanins, Biotropia (2001b) (in press).

[55] VUYLSTEKE, D.R., SWENNEN, R., "Genetic improvement of plantains: the potential of conventional approaches and the interface with in vitro culture and biotechnology", Biotechnology applications for banana and plantain improvement: Proc. of a workshop, San Jose, Costa Rica, 27-31 Jan. 1992. Montpellier, France, INIBAP (1993) 169-176.

[56] Raghavan and Torrey (1964)

[57] ROMBERGER, J.A., TABOR, C.A., The piece abyss shoot apical meristem in culture I. Agar and autoclaving effects, Am. J. Bot. 58 (1971) 131-140.

[58] WIRNICKE, W., KOHLENBACH, H.W., Investigation on liquid culture in nicotine, Z. Pflanzenphysiol. 79 (1976) 189-198.

[59] AFELE, J.C., DE LANGHE, E., Increasing in vitro germination of Musa balbisiana seeds, Plant Cell, Tissue Organ Culture 27 (1991) 33-36.

[60] PLOETZ, R.C., et al., Current status of panama disease in Thailand, Fruits 51 (1997) 387-395.