Abstract: Background and Objective: The most commonly used direct method in breeding triploid banana is crossing tetraploids with diploids. “Pisang Klutuk Sukun” (Musa balbisiana Colla, BB) has been associated with improved vigor and tolerance to biotic and abiotic stresses and therefore, it is a target for Musa breeding program. The objective of the study was to produce banana autotetraploid plants from diploid “Pisang Klutuk Sukun” and to evaluate the morphological characters and the reproductive potential of the autotetraploid plants. Materials and Methods: Induction of the autotetraploid was conducted using in vitro-cultured shoots of diploid “Pisang Klutuk Sukun” treated with oryzalin at a concentration of 60 μM for 7 days in a liquid MS basal medium with addition of 2 mg L–1 BA. The morphology characters of the autotetraploids were evaluated which based on 52 characters of UPOV for two reproductive cycles. The reproductive potential of the autotetraploid plants was conducted by crossing the autotetraploids with a diploid banana cultivar; “Pisang Rejang” to produce triploids. Results: The induction of autotetraploid experiment showed that 12 plants out of 34 plants of oryzalin-treated “Pisang Klutuk Sukun” were autotetraploids. The autotetraploid plants showed drooping leaves instead of erect leaves as in its diploid plants. They had fewer suckers compared to its diploid plants. The fruit diameter (width) of the autoteraploid was larger than those of its diploid. The autotetraploid “Pisang Klutuk Sukun” was successfully crossed with diploid banana cultivar, ‘Pisang Rejang’ and produced triploid hybrids. Conclusion: This study concluded that the banana autotetraploid plants of “Pisang Klutuk Sukun” were produced at a concentration of 60 μM for 7 days in a liquid MS basal medium with addition of 2 mg L–1 BA. The autotetraploids were fertile as well.
INTRODUCTION
Bananas (Musa spp.) are important fruit crop in Indonesia and the world. Indonesia is the center of origin and diversity of cultivated banana1. Based on FAO Statistics, in 2016, Indonesia was the third largest banana producer in the world with a production of about 7.1 Mt. Banana production in Indonesia is threatened by many fungal, bacterial and virus diseases as well as abiotic stress, such as drought. To create new varieties with desired characteristics is quiet challenging. Bananas are difficult to breed. Cross breeding programs are difficult because of the asexual behavior of edible clones, which are sterile and polyploid. High sterility and parthenocarpic nature of most edible bananas as well as a lack of knowledge regarding the type of inheritance of resistance limits success of banana breeding program2. Breeding of most cultivated bananas rely upon conventional sexual hybridization, involving the crossing of triploid cultivars with wild or cultivated diploid parents. Generally, crossing triploid (3x) cultivars with diploid (2x) parents, generates tetraploid (4x) hybrids3. Induced polyploidy in banana plants has been conducted2,4-6. In a research performed, amplification of the leaves and fruits of autotetraploids in comparison to the original diploids were observed4. Likewise, increase in the plant height, number of living leaves at flowering and harvest, pseudo stem diameter and greater fruit and bunch were observed also in autotetraploid “Pisang Lilin”2. Tetraploids are usually induced via mitotic inhibition in vitro. Chromosome doubling of potential diploids allows the production of fertile autotetraploid plants that may be used in crosses with improved diploids to generate secondary triploids (AAA) with disease resistance and fruits with good agronomic characteristics4,7. The availability of 2n gametes is rare and unstable in banana plants and working with autotetraploid plants enables and systematizes the production of triploid hybrids directly from a large number of diploid germplasms4.
“Pisang Klutuk Sukun” (Musa balbisiana Colla, BB) is a diploid local cultivar banana, non-parthenocarpic (seeded), tasty and sweet, medium size banana. It is used for its leaves for wrapping and young fruit for food. The M. balbisiana genome has been associated with improved vigor and tolerance to biotic and abiotic stresses and therefore, it is a target for Musa breeding program8. There are no available reports of tetraploid of “Pisang Klutuk Sukun”. It is important to produce fertile tetraploid “Pisang Klutuk Sukun” to generate secondary triploids which has the best characters of “Pisang Klutuk Sukun”. The present investigation was undertaken to obtain autotetraploid “Pisang Klutuk Sukun” using in vitro shoot treatment with oryzalin, to evaluate the morphological characters of the autotetraploid plants and to assess the reproductive potential of the autotetraploid to generate triploid hybrids.
MATERIALS AND METHODS
Materials: Diploid “Pisang Klutuk Sukun” was accessed from Banana Germplasm Repository, Yogyakarta.
Initiation and multiplication of shoot culture: Initiation and multiplication of shoot culture of diploid “Pisang Klutuk Sukun” was conducted on January-June, 2012. The culture was initiated from shoot tips of banana corm dissected and propagated aseptically on a solid Murashige and Skoog9 (MS) medium as previously described6. Shoot cultures of “Pisang Klutuk Sukun” were established and multiplied in MS medium9 supplemented with 30 g L–1 sucrose, 100 mg L–1 myo inositol, 4 mg L–1 thiamine HCl and 2 mg L–1 BA and solidified by 7 g L–1 agar. The media was adjusted to pH of 5.7-5.8.2. The cultures were maintained at 25°C.
Induction and multiplication of autotetraploid Pisang Klutuk Sukun: Induced tetraploidy was conducted on diploid “Pisang Klutuk Sukun” using oryzalin as described by Van Duren et al.10 with minor modification6. The shoots were extracted from the medium and were treated with the antimitotic agent oryzalin at concentration of 60 μM for 7 days in liquid medium with agitation (60 rpm). After treatment, the shoots were washed three times with distilled water and transferred to a proliferation medium for multiplication. The shoots were then transferred to a rooting medium (MS supplemented with sucrose 30 g L–1 and solidified with 7 g L–1 agar). The explants were kept in a room with a photoperiod of 16 h and a temperature of 25±2°C during their growth phase. The cultures were sub-cultured for 5-6 times to reduce the frequency of mixoploids (plant material containing cells with chromosome number variations) and separate the mixoploids. All of the treated shoots were examined for their ploidy level by using Flow cytometer. Only tetraploid shoots were multiplied for characterization.
The autotetraploid induction and multiplication were conducted in Laboratory of Plant Cell and Tissue Culture of Research Center for Biology, Indonesian Institute of Sciences from June-December, 2012.
Acclimatization and cultivation of the autotetraploids: The plantlets (rooted plants) were transferred to a greenhouse and placed in cultivation pots with a substrate composed of sand, coco peat (coconut fiber) and soil compost (1:1:1) and irrigated under 50% shading. After 60 days, the plants were transplanted to 20 L plastic polybags with the same substrate. After 2 months, banana plants were ready for planted in the field. All treated plants and control were planted in 5 plant rows in a random design and replicated three times.
Identification of ploidy level using flow cytometer: Ploidy detection was performed by using a Partec PAS II flow cytometer (FCM) (Partec GmbH, Munster, Germany). Samples were prepared according to Doležel et al.11 with modification6. Approximately 20-30 mg of fresh leaf samples from cigar leaves (control and treated samples) were chopped with a sharp scalpel blade in a glass Petri dish containing 1 mL of LB01 buffer12 of the following composition: 15 mM TRIS, 2 mM Na2 EDTA, 80 mM KCl, 20 mM NaCl, 0.5 mM spermine, 15 mM mercaptoethanol, 0.1% Triton X-100, pH 7.5. The buffer was supplemented with DAPI (4’, 6-diamidino-2-phenylindole) at final concentration of 2 μg mL–1 to stain nuclear DNA. The suspension of released nuclei was filtered through a 50 μm nylon mesh and kept on ice before analysis. The relative DNA content of the sample was then determined using FCM analysis.
Relative DNA content is given in C units. The 1C value is DNA content of haploid set of chromosomes (n). The distribution of fluorescence intensities (relative DNA content) obtained after flow cytometric analysis is usually in arbitrary units (channel numbers). For ploidy screening, this scale must be calibrated with a reference. In this study, it have used a sample prepared from Musa acuminata ssp. malaccensis (2n = 22) as a diploid reference6 and the flow cytometer was adjusted so that the peak representing its G1 nuclei appeared at channel 200. This setting was kept constant and other samples were characterized by the relative position of their G1 peak.
Evaluation of morphology characters: Morphology characterization was conducted based on 52 characters of International Union for the Protection of New Varieties of Plants13 for two reproduction cycles. The characterization was conducted in Cibinong Science Center, Indonesian Institute of Sciences, from August, 2013-December, 2016.
Crossing for evaluation of the reproductive potential of the autotetraploid bananas: The autotetraploid “Pisang Klutuk Sukun” (BBBB) and diploid “Pisang Rejang” (Musa, AA) were used for reciprocal crosses. The flowers were pollinated and covered with net bags. Seeds were collected from each fruit of the crosses at maturity. The crossings and harvesting were conducted on July-September, 2015. The numbers of hybrid seeds were recorded for each crossing.
Embryo rescue of the hybrid seeds: The hybrid seeds were separated from the pulp by continuous washing in tap water. Washed seeds were transferred to a beaker containing water for 15 min for embryo rescue procedure. Sunken seeds were used, since most of the floating seeds are having no either endosperm and/or embryo. Seed disinfection was performed under sterile conditions in a laminar flow hood. Seeds were treated with 5% sodium hypochlorite for 15 min. Before and after each treatment, the seeds were rinsed with sterile distilled water 2–3 times. Finally, the seeds were transferred to a sterile Petri plate and used for embryo extraction. Embryos were extracted in a chamber under laminar flow. A longitudinal fissure was made in each seed and the whitish, mushroom-shaped embryo was removed. The excised embryos were cultured in medium consisting of Murashige and Skoog9 salts with addition of 2 mg L–1 of 6-benzyl adenine (BA) and the pH was adjusted to 5.8 before auto-claving at 121°C for 20 min. The embryo cultures were kept on dark until shoots were growing. The shoots were then transferred to a media containing a proliferation medium for multiplication. The shoots were then transferred to a rooting medium (MS supplemented with sucrose 30 g L–1 and solidified with 7 g L–1 with a photoperiod of 16 h and a temperature of 25±2°C during their growth phase).
Acclimatization and cultivation of the hybrids: The acclimation and cultivation of the hybrids were conducted similar to those of the autotetraploid plantlets and plants, as mentioned above.
Identification and verification of the hybrids: The hybrids were analyzed for their ploidy by flow cytometry as described above. The confirmation of hybrids was conducted based on DNA profile of ISSR analysis. DNA extraction protocol of Delaporta et al.14 with modification15 and DNA amplification using ISSR marker protocol16,17 were employed for the ISSR analysis. Hybrid identification and cultivation were conducted from January, 2016-June, 2018.
Statistical analysis: Quantitative data of the experiment on evaluation of morphological characters was analyzed for their means and standard deviation on five individuals for three replicates of a completely random design. The data were combined for two cycles of plant reproduction.
RESULTS
Ploidy identification: In this study, flow cytometry was performed on regenerated plants to give an estimation of nuclear DNA content.
| Fig. 1(a-c): | (a) Histogram of diploid control of “Pisang Klutuk Sukun”, (b) Histogram of autotetraploid “Pisang Klutuk Sukun” and (c) Histogram of mixoploid “Pisang Klutuk Sukun” |
Data in Fig. 1 showed the result of flow cytometry measurement with three types of histograms. Control diploid “Pisang Klutuk Sukun” containing 2C DNA showed peak at channel 200 (Fig. 1a), autotetraploid “Pisang Klutuk Sukun” containing 4C DNA showed peak at channel 400 (Fig. 1b) and mixoploid “Pisang Klutuk Sukun” containing 2C and 4C DNA showed peak at channel 200 and 400 (Fig. 1c).
Morphological characteristics of autotetraploid “Pisang Klutuk Sukun”: The 52 morphological characteristics of the autotetraploid and diploid “Pisang Klutuk Sukun” were presented in Table 1. Five morphological characters (numbers of suckers above ground, plant growth habit, compactness of bunch, fruit width and shape of fruit apex) of the autotetraploids were different than those of the diploids.
| Table 1: | Morphology characters of autotetraploid “Pisang Klutuk Sukun” |
| Fig. 2(a-f): | (a) Diploid “Pisang Klutuk Sukun” with upright leaves, (b) Autotetraploid “Pisang Klutuk Sukun” with drooping leaves, (c) Bunch of Diploid “Pisang Klutuk Sukun”, (d) Bunch of autotetraploid “Pisang Klutuk Sukun”, (e) Fruit of Diploid Diploid “Pisang Klutuk Sukun” and (f) Fruit of autotetraploid “Pisang Klutuk Sukun” |
The number of suckers above ground of the autotetraploid was fewer than that of the diploid.
The plant growth habit of the diploid showed upright leaves (Table 1, Fig. 2a), while the autotetraploid exhibited drooping leaves (Table 1, Fig. 2b). The fruit bunch of the diploid were medium (Table 1, Fig. 2c) while the autotetraploids had a compacted fruit bunch (Table 1, Fig. 2d). The fruit width of the autotetraploid was bigger than that of the diploid (Table 1, Fig. 2e, f). The diploid had a truncated fruit apex (Table 1, Fig. 2e), while the autotetraploid has a rounded fruit apex (Fig. 2f).
Crossing for evaluation of the reproductive potential of the autotetraploid bananas: In order to evaluate the reproductive potential of the autotetraploid, autotetraploid “Pisang Klutuk Sukun” were crossed with diploid ‘Pisang Rejang’ to produce triploid hybrids. In this research, from 106 seeds, only 42 seeds had embryos. Twelve out of 20 seedlings were triploids (Table 2).
Flow cytometry was performed on the two parents (i.e., the autotetraploid “Pisang Klutuk Sukun” as a female parent and diploid “Pisang Rejang” as male parent) and the hybrids to give an estimation of nuclear DNA content. Results in Fig. 3 showed the flow cytometry measurement with three types of histograms. The autotetraploid “Pisang Klutuk Sukun” containing 4C DNA showed peak at channel 400 (Fig. 3a), diploid “Pisang Rejang” containing 2C DNA showed peak at channel 200 (Fig. 3b) and triploid hybrid containing 3C DNA showed peak at channel 300 (Fig. 3c).
| Fig. 3(a-c): | (a) Histogram of autotetraploid “Pisang Klutuk Sukun” (female parent), (b) Histogram of diploid “Pisang Rejang” (male parent) and (c) Histogram of triploid hybrid (Autotetraploid “Pisang Klutuk Sukun” x diploid “Pisang Rejang”) |
| Fig. 4: | DNA profile of banana hybrids and their parents (autotetraploid “Pisang Klutuk Sukun” and diploid “Pisang Rejang”) |
M: 100 pb plus DNA ladder (Fermentas), 1, 2: Female parent (Autotetraploid “Pisang Klutuk Sukun”), 3, 4: Male parent (Diploid “Pisang Rejang”), 5, 6: Hybrids |
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| Table 2: | Production of secondary triploid hybrid from autotetaploid “Pisang Klutuk Sukun” x diploid “Pisang Rejang” crosses |
The hybrids were confirmed by DNA profiling using ISSR marker i.e., UBC-811, UBC-815 and UBC-834. Hybrids were identified by the presence of bands of the both parents using three different primers (Fig. 4).
DISCUSSION
In this study, ploidy estimation was conducted by using flow cytometry method. The method was suitable for observing large numbers of samples and for identifying the mixoploids. The ploidy estimation was usually conducted by chromosome counting18. However, this technique was not recommended because the chromosome size of Musa was very small, time consuming and mixoploid was difficult to identified10. Furthermore, this method cannot be used to establish ploidy of non-dividing cells in differentiated tissues, such as leaves. Flow cytometry is used as the method of choice for large-scale ploidy screening in Musa spp. because flow cytometry provides rapidity, convenience and accuracy11.
In this study, the mixoploids were eliminated by sub-culturing until six times, however, the mixoploids were still existed. Mixoploids with different ploidy levels in different plant cells or organs were observed in chromosome5,6 doubling experiment2,12,19-21. Mixoploids may arise because antimitotic agents may not reach all of the meristems on a plant (or those that are actively dividing)12. The ultimate goal was to develop protocols for regeneration of solid autotetraploid plants from mixoploid plants. Roux et al.21 showed that the type of propagation system has an effect on cytochimera dissociation specially when used at an early stage of clonal propagation of mixoploids. Marcotrigiano22 suggested that by destroying the main shoot after one or two weeks post-subculture, a higher proliferation rate could be accomplished and dissociation of chimeras would be more effective.
In this study, all tetraploids were maintained in the field for almost 3 years for evaluation of polyploidy stability; so far, no major change has been seen on the morphological level. The autotetraploid had fewer suckers, drooping leaves and a rounded fruit apex. Similar results were also observed by other researchers2,4-6. Tetraploidy was found to affect fruit size and shape of “Pisang Lilin”2 as well as Mas Jambe”6. Autotetraploid plants had bigger bunch size compared to diploid plants of “Pisang Lilin”2 and “Kluai Leb Mu Nang” and “Kluai Sa”5.
In this study, the autotetraploids produced flowers and could be crossed with diploid and generating triploid hybrids. Bakry et al.4 showed similar results. Induction of banana tetraploid plants is a step to obtain sterile triploid genotypes resulting from a cross between a diploid and a chromosome doubled plant4,14. In the crosses between the autotetraploid and diploid cultivar, the autotetraploid as a male parent (pollen donor) showed fewer developed seeds. On the other hand, when the autotetraploid was used a female parent, many seeds were obtained. Therefore, triploid hybrids were successfully obtained from crossing autotetraploid x diploid, when the autotetraploid were used as female parent. Similar result was also found in the work of Oselebe et al.3.
Although somatic chromosome doubling does not introduce new genetic material and produces only additional copies of existing genes and chromosomes, many genome alterations occur after mitotic polyploidization23. Studies showed that genetic changes often result in polyploid crops being superior to diploids with respect to morphological changes, genetic adaptability and tolerance to environmental stresses24-29. However, the next step will be to evaluate the autotetraploid of “Pisang Klutuk Sukun” to exploit the superiority to vigor, disease resiststance and tolerance to environmental stresses.
CONCLUSION
This study concluded that the banana autotetraploid plants of “ Pisang Klutuk Sukun” (Musa balbisiana, BBBB) were successfully obtained by oryzalin treatment of shoot culture of diploid “Pisang Klutuk Sukun” at a concentration of 60 μM for 7 days in a liquid MS basal medium with addition of 2 mg L–1 BA. The autotetraploids had different morphology characters from their diploid plants, specifically in their drooping leaves, fewer suckers and larger fruit diameter. The autotetraploids were fertile and could be used to produce triploid hybrids.
SIGNIFICANCE STATEMENT
This study discovered the induction of autotetraploid of “Pisang Klutuk Sukun” using oryzalin treatment that can be beneficial for producing 2n gamete for triploid hybrid production. This study revealed that ploidy and morphology of the autotetraploid plants of “Pisang Klutuk Sukun” were stable during two cycles of reproduction. The autotetraploid plants were fertile and can be used a female parent for producing sterile triploid hybrids. This study will help the researchers to uncover the critical areas of providing fertile autotetraploids for producing sterile triploid hybrids that many researchers were not able to explore.
ACKNOWLEDGMENTS
This research was financially supported by the Indonesian Institute of Sciences through Competitive Program of 2012-2016 and DIPA 2016-2018, with grant number No. 900/F/ 2012; No.1000/F/2013, No 934/F/2014; No.3583/IPH.1/HK. 01.03/I/2015; No.544/IPH/HK.01.03/I/ 2016; No.919/IPH/HK. 01.03/I/2017; No.B-669/IPH.1/HK.01.03/I/2018. The authors are grateful to the staffs of the Laboratory of Plant Genetics and Breeding the Laboratory of Plant Cell and Tissue Cultures especially K.Utami Nugraheni SP and Herlina.