African Crop Science Journal African Crop Science Society ISSN: 1021-9730 EISSN: 2072-6589 Vol. 7, Num. 4, 1999, pp. 313-320

African Crop Science Journal, Vol. 7. No. 4, pp. 313-320, 1999

Gamma Rays and Ethylmethane Sulphonate In Vitro Induced Fusarium Wilt Resistant Mutants in Bananas

C. Musoke, P.R. Rubaihayo and M. Magambo
Department of Crop Science, Makerere University, P.O. Box 7062, Kampala, Uganda

Code Number: CS99020

ABSTRACT

Shoot-tips of five different banana and plantain genomes: dessert bananas (Sukalindizi (AB), Gros Michel (AAA), French Plantain (ABB), Kayinja (AAB) and Mbwazirume (AAA-EA) were exposed to ⁶⁰Co gamma irradiation of 20, 30, 40 Gy at a dose rate of 0.8 Gy min^-1. This was followed by different doses for each cultivar as follows: 32, 34, 36, 38 Gy for Gros Michel, 36, 38, 40, 42 Gy for French Plantain, 38, 40, 42 Gy for Kayinja, 32, 34, 36, 38 Gy for Mbwazirume and 27, 30, 32 for Sukalindizi. Radiosensitivity was assessed by determining the growth rate as indicated by fresh weight of cultures 30 days after irradiation. The LD₅₀s for cultivars estimated using relative growth rate and SAS computer programme were found to be cultivar dependent. A similar batch of shoot-tips was exposed to ethylmethane sulphonate (EMS) with concentrations (v/v) of 0.05%, 0.15%, 0.2%, 0.25%, 0.35%, 0.4% and 0.6% for three hours. The LD₅₀s were similarly determined and found to be cultivar dependent. Direct exposure of in vitrobanana plantlets to Oxysporium fsp cubense conidial suspensions suggested that in vitro conditions weakens the resistance of the plantlets to the fungus when compared to older plantlets in the screenhouse.

Key Words: Gamma irradiation, lethal dose (LD₅₀), mutagenesis, Musa

RÉSUMÉ

Les pousses de cinq génomes de banane et plantain: banane de table (Sukalindizi (AB), Gros Michel (AAA), French Plantain (ABB), Kayinja (AAB) et Mbwazirume (AAA-EA) ont été exposées à une irradiation gamma ⁶⁰Co de 20, 30, 40 Gy à une dose de 0.8 Gy par minute. Ceci a été suivi de differentes doses pour chaque cultivar comme suit: 32, 34, 36, 38 Gy pour Gros Michel; 36, 38, 40, 42 Gy pour le French Plantain, 38, 40, 42 Gy pour Kayinja; 32, 34, 36, 38 Gy pour Mbwazirume et 27, 30, 32 pour Sukalindizi: La radioactivité a été évaluée en déterminant le taux de croissance comme indiqué par le poids frais des cultures 30 jours après irradiation. La dose léthale (LD₅₀s) des cultivars estimée par le taux relatif de croissance et un programme du SAS a été trouvée dépendante du cultivar. Un autre lot similaire de gousses a été exposé au sulphonate éthyl de méthane avec des concentrations (v/v) de 0.05%; 0.2%; 0.25%; 0.35%; 0.40% et 0.60% pendant 3 heures. Les doses léthales ont été déterminées de la même manière et ont été trouvées être dépendantes des cultivars. Exposition directe des plantules de banane in vivo aux suspensions conidiales d’ Oxysporium fsp cubense a suggeré que les conditions in vivo affaiblit la résistance des plantules au champignon en comparaison des vieuilles plantules dans la serre.

Mots Clés: Irradiation gamma, dose léthale (LD₅₀), mutagénèse, Musa

Introduction

Bananas (Musa spp.) represent one of the most important crops in the world. In many countries, bananas constitute a major part of the national economy and food security. Research efforts on bananas have focused on development of improved cultivars in terms of agronomic attributes such as yield, disease and pest resistance, among other traits (Purseglove, 1988). However, as reported by Swennen and Vuylsteke (1993), breeding for host resistance in Musa spp., using classical breeding methods is difficult due to loss of good characteristics, high sterility, polyploidity and long generation time. Moreover, many important commercial varieties were excluded from sexual recombination because they were entirely male and female sterile. Rowe (1984) reported that several clones of bananas with good agronomic traits evolved as spontaneous mutations from cultivated clones. However, the frequency of such mutations was so low that induced mutation was suggested to be the main avenue to achieve genetic improvement (Delanghe, 1986; Stover and Buddenhagen, 1986). Both chemical (Hwang and Ko, 1986), and physical (Velez Fortuno and Cedeno Malonado, 1972) mutagens have been used to induce mutations in vivo and would therefore be particularly important for sterile banana cultivars where there is no sexual reproduction, to generate genetic variation. The use of in vitro cultures in mutation breeding of bananas and plantains offers several advantages over the in vivo techniques including obtaining shoot-tips from pre-existing cultures and recovering mutants and rapidly micro-propagating them. To achieve useful in vitro mutations, a suitable exposure of cultures to a mutagen dose that reduces in vitro growth to 50% of the control (50% lethal dose) is needed. Novak (1990) while using gamma irradiation, recommended doses between 30- 40 Gy for triploid cultivars. Omar et al. (1989) achieved LD₅₀ of a diploid SH-3362 clone at 0.2% ethylmethane sulphonate (EMS) after 3 hours of incubations. The work reported in this paper was carried out to assess the 50% lethal doses and behaviour of in vitro shoot-tips of five banana and plantain cultivars after exposure to gamma rays and EMS and the possibility of early detection of banana and plantain mutants resistant to Fusarium wilt.

Materials and Methods

Shoot-tip cubes of about 2 cm³ were isolated from suckers, surface sterilised using 95% ethanol for 20 seconds and later with 10% (v/v) sodium hypochlorite for 20 minutes. Cubes were then rinsed three times in sterile water and aseptically dissected to expose the shoot-tips (5mm) and transferred to Murashige and Skoog medium (1962) modified by Talengera et al. (1994) which had been autoclaved for 15 minutes at 121ºC and pressure of 1.05 kg cm². Three transfers to fresh medium after initiation were carried out before the first subculture. Cultures were maintained at photoperiod of 16 hours at light intensity of 1773+42 Lux and a temperature of 26+2 ºC. Plantlets in third subculture were exposed to the mutagenic agents.

In case of gamma rays, shoot-tips were transferred to petri-dishes containing multi-plication medium and cultures incubated for 2 days before exposure to gamma radiation from a ⁶⁰ Co source. The dose rate of 0.8288 Gy/min was estimated by a fricke dosimeter (O’Donnel, 1970). The fresh weight of five plantlets were taken after 30 days in order to determine response to irradiation. Dosage of 20 Gy, 30 Gy and 40 Gy were initially used to determine effective mutagenic dose for each cultivar estimated from histograms of in vitro growth given by the formula:

Fresh weight of treated shoots
__________________________ x 100
Fresh weight of untreated shoots

Later, the explants were exposed to the following: 32, 34, 36, 38 Gy for Gros Michel, 36, 38, 40, 42 Gy for Plantain; 38, 40, 42 Gy for Kayinja; 32, 34, 36, 38 Gy for Mbwazirume; and 27, 30, 32 Gy for Sukalindizi in order to establish the approximate LD₅₀s. The same data of fresh weight of five plantlets after 30 days were used in a SAS computer programme to facilitate fitting the generalised linear model and estimate the 50% lethal dose. Probability analysis fits a linear regression of the normal deviate corresponding to each proportion P lost, probability (P) and the concentration x (SAS, 1987).

Probability (P) = + log₁₀(conc.)

The procedure calculates maximum likelihood estimates and using the probability link function. The estimates of LD₅₀ were given by setting Probability (0.5) = 0

which implies that Log₁₀ (conc.) = - a /ß
and therefore, LD₅₀ = antilog (- a /ß)

Chemical mutagenesis was carried out by immersing shoot-tips at their third subculture in filter sterilised EMS at concentrations of 0.2 %, 0.4 %, and 0.6% for 3 hours at room temperature (approx. 25ºC) to determine effective mutagenic dose. Immediately after treatment the explants were rinsed three times with sterile water and then transferred onto solid Murashige and Skoog medium (1962) modified by Talengera et al. (1994). Post EMS treatment behaviour was assessed by taking fresh weights of five plantlets after 30 days, and effective dose range estimated as in case of gamma irradiation. On basis of the results of the growth rates shoot-tip cultures in third subculture stage were subjected to doses between 0.05%-0.4% v/v EMS in order to determine LD₅₀s using in vitro growth of cultures 30 days later. The data for in vitro growth were subjected to SAS programme for modelling the proportion of weight loss in response to EMS using the probability link function and 50% lethal doses determined using LD₅₀ = antilog (- a /ß).

Early screening was achieved using Fusarium oxysporium fsp cubense spore population adjusted to 3 x 10⁴ spores ml^-1 using the micro-syringe method. Each plantlet was injected in the corm with 100 µl of the spore suspension and placed on rooting medium. Control plantlets were injected with distilled water. The data were taken on period to first appearance of yellowing of leaves and final death of plantlets. In the screen house, two-months old plantlets were uprooted and roots cut halfway, washed with distilled water and dipped in conidial suspension (3 x 10⁴ spores ml^-1) for 10 minutes. Control plants were dipped in distilled water. The treated plantlets were placed back into the soil substrate and wilted plantlets recorded after a period of 4 weeks.

Results and Discussion

The results of average weight of five in vitro plantlets used to assess growth of shoot-tips treated with gamma irradiation and cultured for 30 days on Talengera et al. (1994) medium are presented in Table 1. There was significant (P< 0.05) reductions in fresh weights and therefore reduced growth in all cultivars with increase in gamma dosage. Mbwazirume (AAA-E.A) showed the most sensitive reaction followed by Bogoya (Gros Michel- AAA). Novak et al. (1987) reported reductions of up to 70% while using clones ‘SH-3142‘ (AA) and ‘SH-3436‘(AAAA) after exposure to doses between 10 Gy and 50 Gy. Among the Acuminata x Balbisiana hybrids, the diploid cultivar Sukalindiizi (AB) was the most sensitive with Kayinja (ABB) and Gonja (French plantain -AAB) expressing similar sensitivity. Novak (1990) reported similar trends which were dependent on ploidy levels while using similar genomes and comparable dosages.

Table 1. Fresh weights of in vitro plantlets cultured for 30 days after irradiation with Gamma rays

The suitable dose ranges for exposure of Musa shoot-tips cultured in vitro were estimated on the basis of 50% growth reductions in reference to the control. The results indicated that the suitable dose ranges for in vitro shoot culture gamma irradiation mutation induction were: 28-32 Gy for Sukalindiizi, 32-36 Gy for Gros Michel, 37-42 Gy for Kayinja, 31-38 Gy for Mbwazirume and 35-43 Gy for Plantain. Further irradiation using the suggested dose was carried out and the results are presented in Figure 1. The 50% lethal doses estimated from Figure 1 are presented in Table 2. The results suggested that the LD₅₀ for Sukalindiizi was achieved at a dose of 31 Gy, Kayinja at 41 Gy, Mbwazirume at 36Gy, Plantain at 42 Gy and Gros Michel at 37 Gy, indicating cultivar dependence.

The same data of fresh weights of five plantlets regenerated from gamma treated shoot-tips at the established dose ranges subjected to SAS analysis and results of the estimated LD₅₀ are also presented in Table 2. The results generated by the SAS programme were also cultivar dependent and in very close proximity to those by the histograms, indicating the reliability of the two methods.

Table 2. The estimated LD₅₀ using the SAS programme and histograms after exposure to gamma irradiations and culturing shoot-tips for 30 days

Means of in vitro growth assessed as fresh weight of five plantlets regenerated from shoot-tips treated with varying concentrations of EMS and cultured for 30 days are presented in Table 3.

Table 3. Fresh weights of in vitro cultures 30 days after treatment with EMS

For all the cultivars, there was a significant (P< 0.05) reduction in fresh weight of plantlets with increased concentration of EMS. Omar et al. (1989) reported reduction in growth of SH- 3362, a diploid Musa acuminata and GN-60 Gy/A, a triploid (AAA) mutant from the Cavendish cultivar ‘Grand Naine’ using 24.69, 49.38, 74.07 or 88.76 mM EMS filter sterilised with 4% dimethyl sulphur oxide (DMSO) for three hours at a constant temperature of 280C.

The histograms of results of cultivar response to 2%, 4% and 6% v/v of EMS doses were used to estimate better exposure doses for determination of LD₅₀ for each cultivar. Subsequently, lower doses were used for estimation of different LD₅₀ for the cultivars and the results are presented in Figure 2. Concentration beyond 0.4% retarded growth in all the cultivars leading to very poor plant vigour and at 0.6% v/v of EMS there was death of all explants except in case of Kayinja. These results demonstrated that chemosensitivity was cultivar dependent. Brunner and Ashri (1986) reported response to chemical mutagens being cultivar specific, and Omar et al. (1989) suggested that this could be attributed to genomic compo-sition and ploidy levels affecting accumulation of mutagenic compounds in the shoot-tips which is an essential step towards mutagen induction.

The results of the estimation of 50% lethal doses from histograms (Fig. 2) and SAS programme are presented in Table 4. The LD₅₀s estimated from Figure 2 and the results of LD₅₀ estimated by the SAS analysis indicated close proximity to each other except for Sukalindiizi where the histogram estimated 0.15 and the SAS estimated half the value of 0.078. The results from the two methods again indicated that any of the methods could reliably be used to estimate the LD₅₀ for EMS with banana and plantain cultivars.

Table 4. The 50% lethal Doses using SAS programme and histograms after exposure to EMS and culturing the shoot-tips for 4 weeks

The results of challenging plantlets with Fusarium oxysporium fsp cubense conidial suspension injected into the corms are presented in Figure 3. The disease progress was significantly (P<0.5) dependent on time, cultivar and their interaction. The cultivar Mbwazirume which is supposed to be resistant to Fusarium oxysporium fsp cubense succumbed to in vitro challenge by the fungus suggesting that the conditions and intensity of the disease challenge broke down the resistance of the cultivar.

Subjecting plantlets at screen house level to challenge by Fusarium oxysporium fsp cubense conidial suspensions led to development of leaf wilting symptoms within 4 weeks after inoculation (Fig. 4). The response to infection was cultivar dependent. The majority of Mbwazirume plants had a score 1 (resistant) with none having a score of 3. Contrastingly, Kayinja which was the most susceptable cultivar had a majority of its plants with scores of 3 (susceptible) and none in the score 1 category.

Conclusions

The results of the study indicated that the LD₅₀ response to both mutagens was cultivar dependent and the induced mutation reduced cultivar susceptability to Fusarium oxysporium fsp cubense. The wilt incidence in the studies on early screening of variants from induced mutations both in vitro and the screen house was found to be cultivar dependent but invitro plantlets succumbed more to Fusarium oxysporium fsp cubense challenge than the older ones in the screen house. The early screening methods for Fusarium oxysporium fsp cubense banana resistance were found to be efficient in identifying susceptable and resistant plantlets at various growth stages.

Acknowledgement

The work reported in this paper was funded by the Rockefeller Foundation Forum Grant RF 96008#9.

References

©1999, African Crop Science Society

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