Development of embryogenic cell suspensions from shoot meristematic tissue in bananas and plantains (Musa spp.)

Plant Science 170 (2006) 104–112 www.elsevier.com/locate/plantsci

Development of embryogenic cell suspensions from shoot meristematic tissue in bananas and plantains (Musa spp.) H. Strosse a,*, H. Schoofs b, B. Panis a, E. Andre a, K. Reyniers a, R. Swennen a a

Laboratory of Tropical Crop Improvement, Department of Biosystems, Division of Crop Biotechnics, K.U.Leuven, Kasteelpark Arenberg 13, 3001 Leuven, Belgium b J. Genotstraat 31, 1080 Sint-Jans-Molenbeek, Belgium Received 17 June 2005; received in revised form 26 July 2005; accepted 10 August 2005 Available online 6 September 2005

Abstract Multiple meristem cultures of 18 varieties belonging to 5 genome types in Musa (AA, AAA, AAA-h, AAB, ABB) were established by culturing elongated shoots on MS medium supplemented with 100 mM BAP. The top layers comprising the most meristematic tissue, i.e. scalps, were excised and induced for embryogenesis on media containing 1–50 mM 2,4-dichlorophenoxyacetic acid (2,4-D). Embryogenic responses were obtained for each of the tested concentrations, with an optimum at 5 mM 2,4-D. From the 24,375 scalps tested, only 3.3% resulted in an embryogenic response. The average embryogenic frequency was 6.0% for cooking bananas (ABB), 3.8% for Cavendish-type bananas (AAA) and 1.8% for plantains (AAB). Once embryogenic complexes were transferred to liquid maintenance medium, embryogenic cell suspensions with high regeneration capacity were obtained. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Embryogenic frequency; Embryogenic cell suspensions; Meristematic tissue (scalp); Musa; Somatic embryogenesis; Plant regeneration

1. Introduction Bananas and plantains are monocotyledonous, perennial herbs, cultivated in nearly 120 countries of the humid and sub humid tropical regions. In the developing world, Musa species are one of the major food sources [1]. Bananas and plantains are, however, prone to many pests and diseases such as fungi, viruses, bacteria, insects, and nematodes [2]. Due to sterility of most important banana and plantain varieties, genetic improvement through conventional breeding is seriously hampered [3,4]. Hence, genetic engineering of bananas and plantains is needed. The choice of candidate tissues for genetic engineering in banana is restricted to meristematic tissue and Abbreviations: BAP, 6-benzylaminopurine; CC, compact embryogenic callus; 2,4-D, 2,4-dichlorophenoxyacetic acid; ECS, embryogenic cell suspension; IAA, indole acetic acid; IC, ideal callus; IE, individual embryos; INIBAP, International Network for the Improvement of Banana and Plantain; NS, number of scalps induced for embryogenesis; PCV, packed cell volume; RD1, embryo regeneration medium; RD2, germination medium; RS, responsive scalps; SCV, settled cell volume; ZZl, liquid embryogenesis maintenance medium; ZZss medium, semisolid embryogenesis induction medium * Corresponding author. Tel.: +32 16 321690; fax: +32 16 321993. E-mail address: [email protected] (H. Strosse). 0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.08.007

embryogenic cell suspensions. The low transformation frequency of explants isolated from multiple meristem cultures indicates that this tissue is not suitable for genetic transformation [5]. In contrast, transformation of embryogenic cell suspensions (ECS) of bananas and plantains is very efficient [5– 7]. ECS are also the material of choice for regenerable protoplast production [8–10] and mutation breeding [11,12]. In bananas and plantains, four procedures exist for the development of embryogenic cell suspensions. They differ mainly in the source of the explant: zygotic embryos [13–14], rhizome slices, and leaf sheaths [15], immature (fe)male flowers [16– 18], and multiple meristem cultures [19–20]. In this paper, we focus on multiple meristem cultures as the starting material from which an approximate 3 mm top layer (i.e. scalp) is excised and cultured on embryogenesis induction medium. The procedure to develop embryogenic cell suspensions derived from shoot meristematic tissue is henceforth referred to as scalp-method. The main advantage of this procedure is that no field access is required, unlike with most of the other methods. As such, virus-indexed plant material can be used and there is no seasonal dependence of the embryogenic response. In this paper, we demonstrate on a large-scale the application of the scalp-method for the establishment of embryogenic cell

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suspensions in a large range of banana and plantain varieties. Also we provide tools to monitor quality control during the entire process. 2. Materials and methods The following subsequent in vitro phases are involved in the scalp-methodology: preparation of multiple meristem cultures, embryogenesis induction, suspension initiation and maintenance, and plant regeneration (Table 1). 2.1. Preparation of multiple meristem cultures The five most important Musa genome types (AA, AAA, AAA-h, AAB and ABB) are represented in our study (Table 2). The genome group of the highland bananas is indicated by AAA-h because this group is clearly distinct from other AAA bananas [21]. Unless otherwise stated, the plant material was received as in vitro multiple (elongated) shoot cultures from the International Network for the Improvement of Banana and Plantain (INIBAP) Transit Centre at K.U.Leuven [22]. Plant material of the wild (seed producing) diploid Calcutta4 (AA group) was obtained through embryo rescue of seeds provided by the International Institute of Tropical Agriculture (IITA), Nigeria. The Cavendish banana Brasilero (provided by the Instituto de Biologia experimental, IBA, Venezuela), Gran enano FHIA (provided by the Honduran Agricultural Research Foundation, Honduras), Gran enano QDPI and Williams QDPI (both provided by the Queensland Department of Primary Industries, Australia) were all supplied as elongated multiple shoots. At the onset of culture, roots from single in vitro shoots or multiple shoots clusters were removed and leaves cut back at 0.5 cm above the apical meristem. Explants were inoculated in 150 ml test tubes on 25 ml standard multiplication medium (p5 medium) consisting of MS basal salts and vitamins [23] supplemented with 10 mM 6-benzylaminopurine (BAP), 1 mM indole acetic acid (IAA), 10 mg/l ascorbic acid, 30 g/l sucrose and solidified with 3 g/l Gelrite1. Before autoclaving at 120 8C for 20 min, the pH of the culture medium was adjusted to 6.2.

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After a culture cycle of 1 month, clusters of the smallest shoots (each containing three to four shoots smaller than 0.5 cm) were selected, cut back and transferred to fresh p5 medium. Shoot cultures were at 27 8C under continuous light of 50 mE m 2 s 1 and a relative humidity of 70% (condition 1). After 2 culture cycles on p5 medium, multiple shoots were grown on p4 medium (as p5 but with 100 mM BAP) under condition 2 (as condition 1 but in darkness). Once a month and before subculturing, the smallest shoots with clusters of meristems at their leaf bases were selected until groups of meristems (about 0.5 cm in diameter) could be cultured separately. Multiple meristem cultures were considered as optimized when the amount of leaf- and corm tissue could not be further reduced in favour of meristematic tissue. The quality of optimised cultures was scored as low (+), moderate (++) or high (+++) depending on the presence of high, medium or low amounts of leaf- and corm tissue between the meristematic domes [20]. 2.2. Embryogenesis induction Scalps (3 mm  3 mm  3 mm to 3 mm  3 mm  5 mm) containing 3–10 meristematic domes were excised from the top layers of optimized multiple meristem cultures (Fig. 1). During the entire embryogenesis induction phase, explants were kept up to 12 months under condition 2 without subculturing. The embryogenesis induction medium ZZss consisted of half strength MS medium, MS vitamins, 10 mg/l ascorbic acid, 1 mM zeatin, 5 mM 2,4-D, 30 g/l sucrose, solidified with 3 g/l Gelrite1. Before autoclaving at 120 8C for 20 min, the pH of the culture medium was adjusted to 6.2. The influence of varying concentrations of 2,4-D (1, 5, 10, 20 and 50 mM) on embryogenesis induction was investigated for varieties belonging to different genome groups [Calcutta4 (AA group), Ingarama (AAA-h group), Gran enano (AAA group), Orishele (AAB group) and Burro cemsa (ABB group)]. Twelve (for 1, 20 or 50 mM 2,4-D) or 132 explants (for 5 and 10 mM 2,4-D) were inoculated per treatment and the entire experiment was performed twice. The number of scalps that were induced for embryogenesis is further referred to as NS.

Table 1 In vitro phases involved, resulting plant material and time needed during the establishment of scalp derived embryogenic cell suspensions (after [19,20] adjusted with current data) Duration (monthsa)

In vitro phase

Resulting plant material

1. Preparation of multiple meristem cultures from which embryogenesis competent explants (scalps) are excised 2. Embryogenesis induction

Optimized multiple meristem cultures (minimal presence of leaf- and corm tissue between meristematic domes)

5–14

Embryogenic complexes (individual embryos, compact and ‘ideal’ embryogenic calli) Embryogenic cell suspensions

3–8

3. Suspension initiation and maintenance Subtotal 4. Regeneration Grand total a

Variety dependent.

3–12 11–34

Rooted plantlets of test tube size

3–8 14–42

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Table 2 Establishment of embryogenic cell suspensions derived from scalps (100 mM BAP) induced for embryogenesis on ZZss (5 mM 2,4-D) in 18 banana and plantain varieties Type (genome), variety Wild diploid (AA) Calcutta4

NS 1937 1368 2406 1296 1872 581 2928 240

Highland (AAA-h) Ingarama Mbwazirume Nyamwihogora

1414 1128 864

Plantain (AAB) Agbagba Obino l’Ewai Orishele

576 336 1685

Cooking (ABB) Burro Cemsa Cacambou Cachaco Dole

756 1833 1804 1351

9.1 6.2a 0.8 0.9 1.5 3.3a 0.8 NR

1.8

NR

0.4

3.3 a

NR NR NR 30.4

4 2 11 11

NR 1.3 0.3 0.9 1.3

NR

17

0.7

0.0 29.0 57.1 35.7 100.0 32.6 0.0

NR NR NR

NR NR 0.6

3.6 10.3 3.7 4.8

NR 33.5

0 18 4 5 4 28 0

NR NR NR

0.5 0.9 2.4 6.0

NR 59

0.1 6.2 0.3 0.6 1.5 3.3 0.4

0.0 0.0 0.0

ECSsuc.est. (%)

ECSini

NR 2.6

0.0

24375

IC (%)

0.0 3.8 a

Cavendish (AAA) Brasilero Gran enano Gran enano FHIA Gran enano QDPI Williams BSJ Williams QDPI Williams

Total

RS (%)

36.4 22.2 30.6 47.8

NR 7 2 2 87

NR 50.0 50.0 40.0 34.1

ECS: embryogenic cell suspensions; ECSini: number of ECS initiated; ECSsuc.est. (%): frequency of successfully established ECS; IC (%): frequency of responsive scalps forming ‘ideal’ callus; NR: not relevant; NS: number of scalps induced for embryogenesis; RS (%): frequency of responsive scalps [embryogenic complexes with individual embryos (IE), compact callus (CC) or ‘ideal’ callus (IC)]. a Underestimate for frequency of responsive scalps since observations for Gran enano and Williams QDPI were focused on ‘ideal’ callus and not on scalps that formed individual embryos or compact embryogenic calli.

Cultures were observed bi-weekly. Six months after inoculation, surviving scalps were classified based on their morphological characteristics: class 1, watery callus or outgrowth of shoot-tips; class 2, globules; class 3, nodular callus; class 4, individual embryos (IE) and/or compact embryogenic complexes

Fig. 1. Development of in vitro multiple meristem cultures in Gran enano (AAA genome). (A) From left to right: multiple shoot cultures after 1 and 2 cycles on standard multiplication medium p5 (10 mM BAP) followed by multiple meristem cultures at 3 and 7 cycles, respectively, on multiplication medium p4 (100 mM BAP) (bar = 1 cm) and (B) scalp isolated from multiple meristem culture (bar = 1 mm).

(CC); class 5, ‘ideal’ callus (IC) (Fig. 2). The term ‘ideal’ calli was only ascribed to homogeneous complexes consisting of numerous tiny transparent embryos originating from a relatively voluminous embryogenic callus (0.5 cm in diameter). Classes 4 and 5 cover the responsive scalps (RS = IE + CC + IC). When different categories of in vitro response were observed on a same explant, only the highest class was taken into account. Per variety and 2,4-D treatment, the frequency of different in vitro responses associated with the aforementioned classes was calculated according to the following formula: in vitro response x (%) = [(number of scalps with in vitro response x)/ NS]  100. Overall embryogenic frequencies [RS (%) and IC (%)] were calculated in the same way, by taking the average of all varieties. Statistical analysis was performed on all responsive scalps (RS) to explore whether the variety induced and various 2,4-D treatments (1–50 mM) had a significant influence on the overall embryogenic frequency. Evaluation of the in vitro response of 6 months old induced cultures gave rise to categorical data (embryogenic response present or not). The SAS 9.11 program was used for the ‘proc-freq’ procedure calculating relative risk estimates. Resulting cohort-values and corresponding 95% confidence limits allowed assessment of significance among the different embryogenic frequencies induced in different

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Fig. 2. In vitro responses associated with induction of embryogenesis in Musa: (A) non-embryogenic nodular callus (bar = 2 mm); (B) embryogenic complex consisting of individual embryos (bar = 0.6 mm); (C) compact embryogenic callus (bar = 2 mm); (D) ‘ideal’ embryogenic callus (bar = 0.6 mm).

varieties and by different 2,4-D concentrations supplemented to the ZZss medium. The varieties and corresponding number of scalps that were induced for embryogenesis (NS) on ZZss medium (5 mM 2,4D) are listed in Table 2. Induced cultures were observed at least once a month during 1 year after induction. For the majority of induced cultures, all responsive scalps (RS) have been counted and embryogenic frequencies [RS (%) and IC (%)] calculated as before. For two Cavendish varieties (Gran enano and Williams QDPI) observations were focused on the development of ‘ideal’ calli. As such, numbers and consequently also frequencies of responsive scalps for these two varieties have to be considered as underestimates. 2.3. Initiation and maintenance of embryogenic cell suspensions Embryogenic calli were isolated from the parental tissue and transferred into liquid ZZl medium (as ZZss but without gelling agent). The number of suspensions initiated (ECSini) per variety is indicated in Table 2. Cell suspensions were preferably initiated from ‘ideal’ embryogenic calli. In case only heterogeneous embryogenic complexes were available, large embryos (length exceeding 0.5 mm) and compact structures were removed from the embryogenic complex while remaining embryogenic callus and very small embryos (less than 0.2 mm in length) served as inoculum. One embryogenic complex was initiated per 25 ml Erlenmeyer flask with 5 ml ZZl medium. Erlenmeyer flasks with suspensions were sealed with aluminium foil, Parafilm1 and subsequently cultivated on a rotary shaker at 70–90 rpm under conditions 2. During the first 3 months after initiation, 50% of the culture medium was refreshed every 7–10 days; from approximately the third month after initiation, the culture medium was refreshed every 14 days keeping 10–20% of the old ‘preconditioned’ medium. Yellowish round shaped globules, necrotic tissue, browning embryos and large cell clumps (diameter exceeding 0.5 mm) were removed with a plastic bulb pipet. The settled cell volume (SCV) at the onset of a subculture period was adjusted to 1.5– 3%. Larger containers containing 10–60 ml suspension (in 50– 250 ml Erlenmeyer flasks) were used when the SCV exceeded 3%. Besides observation of liquid cultures under an inverse binocular microscope at the end of each subculture period, samples of embryogenic cell suspensions were regenerated monthly to check their quality (see below). Suspensions were considered as successfully established when they were

characterized by a high proportion (>80%) of embryogenic cell aggregates, a multiplication ratio between 1.5 and 2 per 2week subculture period and a high regeneration capacity (according to the qualitative regeneration test described below) during at least 5 months. The frequency of successfully established cell suspensions was calculated according to the formula: ECSsuc.est. (%) = (number of successfully established ECS/total number of ECS initiated)  100. 2.4. Embryo and plant regeneration A qualitative regeneration test was performed monthly for all suspensions under establishment. After homogenisation of the cell suspensions by slightly shaking, 500 ml of the cell culture was spread onto regeneration medium RD1 (13 ml in a Petri dish of 45 mm diameter). The composition of the regeneration medium RD1 was the same as the semi-solid ZZss embryogenesis induction medium, without plant growth regulators and supplemented with 100 mg/l myoinositol. Petri dishes with regenerating cell cultures were incubated under condition 2. Six to eight weeks after inoculation, the embryogenic capacity of cell suspensions was scored by estimating the percentage of surface of the plated sample covered by embryos—score 0: no survival, 0%; score 1: growth of non-embryogenic callus, 0%; score 2: low, sporadic growth of embryos, >0 to <33%; score 3: moderate, >33 to < 75%; score 4: high, >75–100%. We evaluated as such 1279 individual regeneration events (i.e. Petri dishes). The age of tested suspension samples (AAA, AAB, ABB genome) ranged from 1 month to 1.5 years after initiation. The frequency of regeneration scores was calculated according to the formula: frequency score x (%) = (number of individual regeneration events resulting in score x/ 1279)  100. A quantitative regeneration method was performed for established cell suspensions of Cavendish banana (AAA group) and plantains (AAB group). Based on weight measurements and quantity of germinating embryos and plants, the regeneration capacity of cell suspensions per millilitre of settled cell volume was determined [24]. 3. Results The different in vitro phases involved in the scalpmethodology, the resulting plant material and the duration of the different steps are indicated in Table 1.

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3.1. Preparation of multiple meristem cultures Rapidly multiplying meristem cultures could be prepared for all 18 varieties. In vitro proliferation of meristems was stimulated by the application of an elevated BAP concentration in combination with frequent subculturing (Fig. 1). Two culture cycles on p5 medium (10 mM BAP) were sufficient to reduce outgrowth of elongated shoots and to induce the formation of multiple stunted shoots. Yet, in these heterogeneous multiple shoot cultures, small (sometimes even rooted) shoots remained present. A further increase of the BAP content to 100 mM in the culture medium was required to obtain more homogeneous cultures composed of meristematic tissue and less than 10% of undesired differentiated (corm and leaf) structures (Fig. 1). The number of monthly p4 culture cycles required for a maximal reduction of corm and leaf tissue varied between as well as within different genome groups (Table 3). Optimized meristem cultures of the cooking banana (ABB group), plantains (AAB group) and Cavendish banana (AAA group) were obtained after, respectively, 5–9, 7–11 and 8–10 monthly cycles on p4 medium. These multiple meristem cultures, most suitable for scalp excision, were characterized by groups of tightly packed meristems on bulbous (ABB group) or domelike (AAB and AAA group) structures. Outgrowth of roots and shoots could also be suppressed for highland bananas (AAA-h group) and the diploid Calcutta4 (AA group). However, meristematic tissues of these varieties remained rather individually arranged and interspersed by corm and small leaves, even after 9–14 subsequent culture cycles on p4 medium. As such, high-quality AAA-h and AA multiple meristem cultures (like with the ABB, AAB and ABB varieties) could never be obtained.

During the first weeks after inoculation, the presence of 2,4D in the culture medium caused a dedifferentiation into watery callus of the leaf tissue surrounding the meristems. The formation of this watery callus was most ample at the lowest 2,4-D concentration (1 mM). Approximately 1 month after inoculation, yellow and white meristematic globules appeared at the scalp surface. The formation of round shaped meristematic globules was most often followed by the development of kidney shaped heterogeneous globules and non-embryogenic nodular callus. These were observed at each of the 2,4-D concentrations tested. The frequency at which nonembryogenic nodular callus was formed, was in general positively correlated with the amount of leaf tissue present in the initial explant and negatively correlated with increasing 2,4D concentration. Only for Ingarama (AAA-h group) and Calcutta4 (AA group), a 2,4-D concentration of at least 5 mM was required to induce nodular calli. Globules and nonembryogenic secondary calli of all Musa varieties turned brown 4–8 months after embryogenesis induction (data not shown). An embryogenic response was observed at 3–8 months after embryogenesis induction and consisted in the formation of individual embryos, compact callus and/or friable embryogenic callus bearing numerous translucent proembryos (‘ideal’ embryogenic callus). The embryogenic frequency of scalps [(IE + CC) (%) and (IC) (%)] of each Musa variety treated with 5 or 10 mM 2,4-D is shown in Fig. 3A and B, respectively. Both the nature and frequency of embryogenic response depended on the variety and 2,4-D concentration. While no embryogenic response could be observed for Ingarama (AAA-h group) and

3.2. Embryogenesis induction The following in vitro responses were distinguished during the application of a broad range of 2,4-D concentrations (1, 5, 10, 20 and 50 mM) in the embryogenesis induction medium: watery callus or outgrowth of shoot tips (class 1), globule formation (class 2), nodular callus (class 3, Fig. 2A), individual embryos (IE) and/or compact embryogenic complexes (CC) (both class 4, Fig. 2B and C, respectively) and ‘ideal’ callus (class 5, Fig. 2D). Table 3 Number of monthly subculture cycles (on p4 medium) required for the optimisation of Musa multiple meristem cultures and suitability for excising scalps according to the genome type of tested varieties Genome AA AAA-h AAA AAB ABB

Number of subculture cycles

Suitability for scalp excision a

9–14 10–12 8–10 7–11 5–9

+/++ +/++ +++ +++ +++

a Quality high, +++; moderate, ++; low, + (the less the presence of corm and leaf tissue, the higher the quality).

Fig. 3. Embryogenic response 6 months after inducton of scalps of 5 Musa varieties [Calcutta4 (Ca), Gran enano (GE), Ingarama (Ing), Orishele (Ori), Burro Cemsa (Bc)] on ZZss embryogenesis induction medium supplemented with 2,4-D (A) at 5 mM and (B) at 10 mM. White and black bars represent the frequency of embryogenic response consisting of either individual embryos (IE) or compact embryogenic complexes (CC) and ‘ideal’ callus (IC), respectively. For each variety, 132 explants (scalps) were induced at both 5 and 10 mM 2,4-D. Data are averages from 2 independent experiments. In both graphs, different letters above bars indicate significant different results (P < 0.05).

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Fig. 4. Embryogenic response 6 months after inducton of scalps of 5 Musa varieties [Calcutta4 (Ca), Gran enano (GE), Ingarama (Ing), Orishele (Ori), Burro Cemsa (Bc)] on ZZss embryogenesis induction medium supplemented with 1–50 mM 2,4-D (NS = 12 at 1, 20 and 50 mM, respectively; NS = 132 at 5 and 10 mM 2,4-D, respectively). Data on embryogenic frequencies are calculated disregarding the Musa variety tested. White bars represent the frequency of in vitro response consisting of either individual embryos, compact embryogenic complexes or ‘ideal’ callus. Black bars represent the frequency of only ‘ideal’ callus. Different letters above bars indicate significant different results (P < 0.05). Data are averages from two independent experiments.

Calcutta4 (AA group), individual embryos developed on meristematic tissue of Gran enano (AAA group) (at each 2,4-D concentration), Burro cemsa (ABB group) (at each 2,4-D concentration) and Orishele (AAB group) (2,4-D concentration ranging from 1 to 10 mM) (data partly shown). Categorical data analysis of embryogenic frequencies calculated respective to the variety induced at 5 mM 2,4-D, indicated a significant higher embryogenic response of Gran enano compared to Burro cemsa and Orishele. This significant influence on the embryogenic response vanished when shoot meristematic tissue of responsive varieties was induced at 10 mM 2,4-D. The formation of ‘ideal’ embryogenic callus was restricted to Gran enano (24 and 6% of induced explants at 5 and 10 mM 2,4-D, respectively) and Orishele (2 and 9% of induced explants at 5 and 10 mM 2,4-D, respectively). All Burro cemsa explants survived the 2,4-D concentrations tested (even up to 50 mM). For Ingarama, Calcutta4 and Orishele the amount of surviving explants was already considerably reduced (to less than halve of the total number of induced explants) at 20 mM 2,4-D while treatment with 50 mM 2,4-D was completely detrimental (data not shown). Categorical data analysis of embryogenic frequencies calculated irrespective of the variety tested, revealed a significant advantageous effect of inducing meristematic tissue (scalps) at 5 mM 2,4-D compared to other 2,4-D treatments

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(Fig. 4). As such, 5 mM 2,4-D has been selected for the standard embryogenesis induction medium for any banana and plantain variety. As deduced from Table 2, an embryogenic response was obtained on average for 3.3% of all (NS = 24,375) induced scalps. ‘Ideal’ callus was formed on only 1.3% of induced explants. Unlike for the diploid Calcutta4 and highland bananas Ingarama, Mbwazirume and Nyamwihogora, embryogenesis was successfully induced for all other 14 varieties belonging to different genome types. The highest embryogenic frequencies were obtained for the cooking (ABB group) and Cavendish bananas (AAA group) (6.0 and 3.8%, respectively) while this was only 1.8 % in case of plantain scalps (AAB group). The formation of ‘ideal’ callus was characteristic for the Cavendish bananas [IC of 2.6% versus 0.4 and 0.7% for the AAB and ABB group, respectively). However, the embryogenic frequency was not only dependent on the genome group, but it also varied with the variety within genome groups and even from one experiment to another. For example, for the variety Williams QDPI (Cavendish banana), embryogenic responses of 0–22.2% were obtained depending on the experiment (data not shown). 3.3. Initiation and maintenance of embryogenic cell suspensions Success rates for the initiation of good quality embryogenic cell suspensions depended largely on the quality of the selected embryogenic complex. Compact embryogenic calli were not suitable for suspension initiation since they did not disperse into embryogenic cell groups. Large embryos (length exceeding 0.5 cm) either turned black and ceased releasing embryogenic cell material or dedifferentiated into globules that only released non-embryogenic cells. As such, ‘ideal’ complexes for transfer to liquid medium consisted of embryogenic callus and earlystage transparent embryos. Besides its nature, also the size of the embryogenic complex played an important role in the establishment of ECS. Generally, an embryogenic callus clump of 3 mm in diameter was inoculated in 5 ml of liquid ZZ medium. However, due to the rather limited availability of ‘ideal’ calli, a real optimal initial inoculum density could not be determined. Although ‘ideal’ calli were preferably used for suspension initiation, the freshly inoculated cell suspensions remained very heterogeneous during the first months after their initiation (Fig. 5A). Small embryogenic cell aggregates were released

Fig. 5. Embryogenic cell suspensions and regenerating cultures in Musa: (A) heterogeneous cell suspension 2 months after initiation (bar = 193 mm); (B) embryogenic cell suspension 8 months after initiation (bar = 193 mm); (C) development of embryos (bar = 0.5 cm); (D) germination of embryos (bar = 1.1 cm).

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either directly from the embryogenic complex (in case of ‘ideal callus), at the surface of heterogeneous globules, or near the base of small transparent embryos. The quality of the embryogenic cell suspension was negatively influenced by the presence of yellowish round shaped meristematic globules that released non-regenerable empty and/or dense starchy cells. Also, the presence of whitish cotyledonary staged embryos was unfavourable since they dedifferentiated into meristematic globules and/or released phenolic compounds that oxidized and subsequently caused blackening throughout the entire cell suspension. Refreshment of the culture medium and removal of non-embryogenic structures every 7–10 days resulted in a gradual increase of the proportion of embryogenic cell aggregates (Fig. 5B). Growth rate of the cell suspensions varied highly from one suspension to another but was initially very low. In general, 6 months old cell cultures had reached a volume of 1–2 ml settled cells (250 ml Erlenmeyer flask containing 60 ml cell suspension). Suspensions were successfully established at a frequency of 33.5% (59 out of 176), 30.4% (17 out of 56) and 47.8% (11 out of 23) for Cavendish banana (AAA group), plantains (AAB group) and cooking banana (ABB group), respectively (Table 2). On average 34.1% embryogenic calli transferred to liquid medium gave successfully rise to established embryogenic cell suspensions. 3.4. Embryo and plant regeneration The embryo regeneration pattern of 8 weeks old cultures reflected the composition of the cell suspension prior to transfer onto RD1 medium. Embryogenic cell aggregates gave rise to a mixture of globular to polar shaped embryos (Fig. 5C). Inoculated white, round shaped and dense cells developed into non-regenerable white callus. Highly vacuolated cells turned black without further development. The frequency distribution of different regeneration scores ascribed to Musa cell cultures is shown in Fig. 6. Up to 1.5 years after initiation, the majority of ECS (87%) has retained its embryogenic potential. For about one third (37%) of the tested ECS, the plated surface regenerating into embryos even exceeded 75%. Two months after transfer of mature embryos onto germination medium

Fig. 6. Frequency distribution of different regeneration patterns (0: no growth; 1: growth of non-embryogenic callus; 2: sporadic growth of embryos; 3: 33– 75% of the plated surface is regenerating into embryos; 4: 76–100% of the plated surface is regenerating into embryos) ascribed to Musa embryogenic cell suspensions (AAA, AAB or ABB genome) tested less than 1.5 year after initiation. Regeneration patterns were evaluated on 8 weeks old RD1 cultures. In total 1279 individual regeneration events were performed.

RD2, small (rooted) shoots (Fig. 5D) could be isolated and grown on standard plant regeneration medium (REG). Quantitative embryo regeneration tests showed that plantain (AAB group) and Cavendish (AAA group) cell suspensions yielded 3.6  104 to 4.7  105 and 1.5  105 to 1.8  105 embryos per ml settled cells, respectively. Highest embryoplant conversion frequencies noted for Cavendish bananas resulted in higher average plant regeneration frequencies compared to plantains (4.8  104 and 2.2  104 plants per ml settled cells for Cavendish and plantains, respectively). 4. Discussion This paper presents for the first time both a qualitative and quantitative account of the establishment of Musa embryogenic cell suspensions derived from shoot meristematic tissue. It involved large-scale experiments with 24,375 explants of 18 varieties classified into 5 genome types. As such, we were able to demonstrate that the scalp-method is applicable to a wide range of banana and plantain varieties. Unlike most dicotyledoneous plants [25] and seed setting monocots [26], we found that the establishment of a plant regeneration system for Musa spp. via somatic embryogenesis remains time consuming (14– 42 months), labour intensive and rather inefficient. A first bottleneck in the establishment of Musa embryogenic cell suspensions is the availability of embryogenesis-competent explants. For immature zygotic embryos, secondary embryogenesis could be induced [13,14] and cell suspensions established [27]. However, in Musa spp. this method is restricted to seed setting varieties only, thus excluding edible varieties with parthenocarpic fruits. In 1989, Novak et al. [15] reported on cell suspensions prepared from rhizome callus. To our knowledge, this protocol could not be repeated elsewhere. As described by the team of Ganapathi et al. [28], suspensions derived from generally available thin shoot-tip sections yielded only 200 regenerants per 0.5 ml PCV making this system rather unsuitable for genetic engineering. The well-documented male flower method [16,17, 29–31] is applicable to most Musa varieties provided immature male flowers are available, thus excluding most of the preferred plantain types. The female flower method, however, is applicable to any variety but leads to the destruction of entire bunches [18]. Besides the seasonal dependent embryogenic response of initial explants, the (fe)male flower method relies on direct field access, which is a major limitation. The major drawback of the reported scalp-method is the lengthy material preparation phase. In contrast to other monocots-like maize [32] and oat [33] for which embryogenesis competent multiple shoot cultures can be obtained within a few weeks, the material preparation phase in banana remains extensive and takes 5–14 months. Although embryogenesis is triggered at different 2,4-D concentrations for the male flower- and scalp-method (respectively, 18 and 5 mM), both methods are rather similar once embryogenesis is induced. Escalant et al. [29] and Grapin et al. [18] also report on low and variable embryogenic frequencies with the ‘ideal’ callus formed in at least 3 months

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old induced cultures (on average 8% of male flower buds giving rise to an ‘ideal’ callus when about 8 immature male flower hands are inoculated per bud). Success rates in the establishment of Musa ECS and morphological characteristics of cell cultures derived from multiple meristem cultures are in accordance with results obtained via the male flower technology [16,24,30]. A strong correlation exists between the composition of the scalp-derived ECS and its embryogenic potential as evaluated by our fast and easy qualitative embryo regeneration test. Although this regeneration test is undoubtedly suitable for a first quality screening of the established suspension, comparison of regeneration capacity between cell cultures requires application of a more standardized, precise and labour intensive quantitative regeneration method. Coˆte et al. [16] reported for the Musa AAA variety Grande naine 3–20% germination of 3.7  105 embryos per ml packed cell volume (PCV), while embryogenic cell suspensions of the plantains French Sombre and Curare enano (both AAB group) yielded 105 and 105 to 5  105 embryos per ml PCV, respectively. The embryogenic cell suspensions we established for different varieties belonging to different genome types, were characterized by a comparable high embryo regeneration (1.5  105 to 4.7  105 embryos per ml settled cells) and germination frequency (2.2  104 to 4.8  104 plants per ml settled cells). Besides the important time aspect, the efficiency of a plant regeneration system through cell suspension cultures is determined by following subsequent key factors: (i) the availability of embryogenesis-competent explants, (ii) the nature and frequency of embryogenic response, (iii) the success rate of suspension initiation and (iv) the plant regeneration frequency. Extreme high embryogenic frequencies (90–100% embryogenic callus) were obtained in cassava [34,35] and the ornamental bleeding heart [36]. Low to moderate embryogenic frequencies (<50%) could be compensated by a fast development of the embryogenic callus (3–4 weeks in Bermuda grass [37]) or relatively fast establishment of embryogenic cell suspension from callus (2 months for oil palm [38]). Compared to other plant species, banana tissues are extremely recalcitrant with respect to embryogenic response. The limited choice of explants, the lengthy material preparation phase, the low and variable embryogenic responses and time consuming quality improvement of liquid cultures involved in the scalp-methodology imply that the establishment of Musa embryogenic cell suspensions needs further improvement. This is unquestionably the case for the varieties Calcutta4 and all highland bananas where until now, embryogenic cell suspensions were never obtained. Once established, these cell suspensions can be subject to somaclonal variation and microbial contamination and a prolonged culture period may result in total loss of morphogenic capacity. Cryopreservation therefore is an essential tool for the safe storage of established ECS. Hence, all cell lines successfully established as a result of this study are currently safely stored in liquid nitrogen using the method of Panis et al. [39]. Also some of these suspensions are now part of a genetic engineering program for the production of fungal

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resistant bananas and plantains [40,41] and the search of banana and plantain genes [42]. Acknowledgements This work was possible due to the financial support of DGIC (Directorate-General for International Cooperation), Belgium through a grant to IITA (International Institute of Tropical Agriculture) and INIBAP (International Network for the Improvement of Banana and Plantain). This research falls within the strategy of ProMusa. We thank F. Coˆte and R. Domergue of CIRAD (Centre de Coope´ration Internationale en Recherce Agronomique pour le De´veloppement) for the fruitful discussions on somatic embryogenesis in Musa. References [1] E. Frison, S. Sharrock, The economical, social and nutritional importance of banana in the world, in: C. Picq, E. Foure´, E. Frison (Eds.), Bananas and Food Security, Proceedings of the International Symposium on Bananas and Food Security, Douala, Cameroun, 1998, pp. 21–35. [2] D.R. Jones, Diseases of Banana, Abaca´ and Enset, CABI Publishing, Wallingford, UK, 2000. [3] R. Swennen, D. Vuylsteke, Banana (Musa L.), in: R. Raemaekers (Ed.), Crop Production in Tropical Africa, DGIC (Directorate General for International Cooperation), Brussels, Belgium, 2001, pp. 530–552. [4] A. Tenkouano, R. Swennen, Progress in breeding and delivering improved plantain and banana to African farmers, Chronica Horticult. 44 (2004) 9– 15. [5] R. Swennen, G. Arinaitwe, B.P.A. Cammue, I. Franc¸ois, B. Panis, S. Remy, L. Sa´gi, E. Santos, H. Strosse, I. Van den houwe, Transgenic approaches for resistance to Mycosphaerella leaf spot diseases in Musa spp., in: L. Jacome, P. Lepoivre, D. Marin, R. Ortiz, R. Romero, J.V. Escalant (Eds.), Mycosphaerella Leaf Spot Diseases of Banana: Present Status and Outlook, Proceedings of the Workshop on Mycosphaerella Leaf Spot Diseases San Jose´, Costa Rica, 2002, pp. 209–238. [6] L. Sa´gi, B. Panis, S. Remy, H. Schoofs, K. De Smet, R. Swennen, B.P.A. Cammue, Genetic transformation of banana and plantain (Musa spp.) via particle bombardment, Biotechnology 13 (1995) 481–485. [7] L. Sa´gi, S. Remy, B.P.A. Cammue, K. Maes, T. Raemaekers, B. Panis, H. Schoofs, R. Swennen, Production of transgenic banana and plantain, Acta Horticult. 540 (2000) 203–206. [8] B. Panis, A. Van Wauwe, R. Swennen, Plant regeneration through direct somatic embryogenesis from protoplasts of banana (Musa spp.), Plant Cell Rep. 12 (1993) 403–407. [9] R. Megia, R. Haı¨cour, S. Tizroutine, V. Bui Trang, L. Rossignol, D. Sihachakr, J. Schwendiman, Plant regeneration from cultured protoplasts of the cooking banana cv. Bluggoe (Musa spp., ABB group), Plant Cell Rep. 13 (1993) 41–44. [10] R. Haı¨cour, A. Assani, K. Matsumoto, A. Guedira, Banana protoplasts, in: S. Mohan Jain, R. Swennen (Eds.), Banana Improvement: Cellular, Molecular Biology, and Induced Mutations, Science Publishers Inc., Enfield, NH, USA, 2004, pp. 111–125. [11] N. Roux, A. Toloza, J.P. Busogoro, B. Panis, H. Strosse, P. Lepoivre, R. Swennen, F.J. Zapata-Arias, Mutagenesis and somaclonal variation to develop new resistance to Mycosphaerella leaf spot diseases, in: L. Jacome, P. Lepoivre, D. Marin, R. Ortiz, R. Romero, J.V. Escalant (Eds.), Mycosphaerella Leaf Spot Diseases of Banana: Present Status and Outlook, Proceedings of the Workshop on Mycosphaerella Leaf Spot Diseases San Jose´, Costa Rica, 2002, pp. 239–250. [12] N. Roux, Mutation induction in Musa—review, in: S. Mohan Jain, R. Swennen (Eds.), Banana Improvement: Cellular, Molecular Biology, and Induced Mutations, Science Publishers Inc., Enfield, NH, USA, 2004, pp. 23–32.

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