Indian Journal of Biotechnology Vol 16, January 2017, pp 119-125 Plant regeneration through isolated microspore culture in recalcitrant durum wheat genotypes Yasemin Coskun* and Cigdem Savaskan Department of Biology, Faculty of Arts and Sciences, Suleyman Demirel University, Isparta 32 260, Turkey Received 10 June 2015; revised 28 December 2015; accepted 2 January 2016 In the present study, a protocol of isolated microspore culture was optimized to regenerate green plants in Turkish durum wheat genotypes (Kiziltan-91, C-1252, Mirzabey 2000 & Kunduru-1149). The bread wheat cultivar (Gun-91) was used as control because of its high androgenic response in the microspore culture. First significant step was to treat the anthers with four pretreatments (cold, cold with mannitol, cold with sorbitol & mannitol at room temperature). maceration was used as an isolation method and microspores were plated on induction culture medium supplemented with arabinogalactan-proteins (AGP) and ovary coculture. When the embryos reached the size of 2 mm, they were transferred to the differentiation medium having a combination of phenylacetic acid (PAA) and gibberellic acid (GA 3 ). The best results were obtained with the pretreatment of mannitol (+4 C) for 7 d on providing embryos and regenerated green plants in four durum wheat genotypes. The cultivar Kiziltan-91 gave the best response for embryo (>2 mm) formation (20.63%) and cultivar Kunduru-1149 for green and total plant regeneration (6.25% and 18.75%). These results indicated that the present protocol performed well to increase the embryo formation and green plant regeneration in the recalcitrant durum wheat genotypes studied. Keywords: Durum wheat, genotype, mannitol, microspore culture, pretreatment. Introduction Microspore culture is one of the most powerful methods to produce haploids or doubled haploids (DH) in cereals by androgenesis. DH technology generates homozygous lines very fast, which can be used to obtain a product with desired traits 1. However, significant complications still occur in providing green plant regeneration in durum wheat (Triticum durum Desf.) genotypes. Two important problems of microspore culture of durum wheat are needed to be focused before it can be used for breeding programmes. Variation in the behavior of genotypes in microspore culture is the first and regenerated high proportion of albino plantlets is the second problem 2. The most important step to obtain good regeneration of embryos through microspore culture is to trigger the microspores to a sporophytic pathway. Various pretreatments are used to induce stress in cereal species, which switch microspores from gametophytic programme to the *Author for correspondence: Tel: +902462114154 yascoskun@gmail.com sporophytic pathway 3. In bread wheat (T. aestivum L.), pretreatment of spikes with cold or heat shock, chemicals, carbohydrate starvation, water stress, radiation etc acts as an external stimulus to trigger or enhance microspore embryogenesis. These factors may act alone or in combination in order to achieve an optimal conversion of microspores to embryogenic cells 4,5. To obtain a worldwide method of microspore culture for all wheat genotypes is an impossible task because of the differences in embryogenic response amongst themselves. General protocol of isolated microspore culture includes the following basic steps: growing of donor plants, anther collection, pretreatments, isolation of microspores, culture medium, induction of microspores to regenerate embryos 1. In contrast to hexaploid bread wheat, few studies on microspore embryogenesis have been reported in tetraploid durum wheat. Because of its recalcitrant nature to microspore culture, most of the cultivars did not regenerate green plants with all procedures 6. These problems have to be resolved in order to use durum wheat cultivars in microspore culture technique and applications in wheat biotechnology.
120 INDIAN J BIOTECHNOL, JANUARY 2017 In the present study, four different Turkish durum wheat genotypes (tetraploid-aabb, 2n=4 =28) have been investigated to find out an efficient protocol for embryo production and green plant regeneration through microspore culture technique. Materials and Methods Plant Material Four durum wheat cultivars, viz., Kiziltan-91, C-1252, Mirzabey 2000 and Kunduru-1149, were used as plant material. Bread wheat (hexaploid- AABBDD) cv. Gun-91 (2n=6 =42) was used as control because of its high androgenic response in microspore culture. Seeds were vernalized for 1 month at 4 C and then were placed in pots with a mixture of peat and sand (3:1). Each pot contained two to three plants after the germination of seeds. Plants were kept in a growth room at 24±2 C temperature during the day and 18±2 C during the night, with a 16 h photoperiod, 51-54% humidity and 12,000 lux light intensity. Pretreatment Spikes were harvested from the pot grown plants when the majority of microspores were in the mid to late uninucleate stage. Microspores were stained in acetocarmine to see the stage of microspores based on the location of the nucleus relative to the microspore pore. Collected tillers were stored in water at 4 C up to 4 d, until sufficient spikes (5-10) were collected. For sterilization, spikes were removed from their sheaths in a laminar flow bench and sprayed with 70% ethanol. Spikes were plated on four different pre-treatments (Table 1): (i) cold (+4 C), (ii) 0.4 M Table 1 Pretreatment conditions used for wheat genotypes No. Material Pretreatment Conditions (i) Spike 0.5 ml sterile water +4 C, 7 d, dark (ii) Spike partially immersed +4 C, 7 d, dark (iii) Spike 0.4 M sorbitol partially immersed 0.4 M sorbitol +4 C, 7 d, dark (iv) Spike partially immersed 5 d, dark mannitol (+4 C), (iii) 0.4 M sorbitol (+4 C) for 7 d, and (iv) (room temperature) for 5 d in the dark. s from flowers on the central part of each spike of pretreatment (i), (ii) and (iv) were then placed in solution and stored for 1 d at room temperature (~24 C) in the dark. Only the anthers of pretreatment (iii) was placed in 0.4 M sorbitol solution and stored under the same conditions. Isolation of Microspores After pretreatment, the microspores already shed into mannitol solution were transferred to 10 ml centrifuge tube, while the microspores still remaining in anthers were isolated in 0.4 M mannitol by filtration and added to the centrifuge tube. For isolation, the anthers in mannitol solution were placed in Millipore filter system with 100 µm nylon mesh and squeezed with a glass rod. The solution on the filter paper was collected under the filter system by a vacuum pump. The microspores both shed and isolated in mannitol (0.4 M) were then centrifuged at 3500 rpm for 5 min and filtered through a 41 µm nylon mesh. Thus, the anther wall debris was completely discarded. After 2-3 washes in by centrifugation for 5 min at 3500 rpm, collected microspores were counted under a microscope with a hemacytometer. For culture, microspore density was adjusted to approx 50,000-100,000 microspores per plate. A similar procedure was applied in case of pretreatment (iii). Culture Media The MMS4 7, a modified MS medium 8, was used as induction medium. The medium differences to MS include: lower inorganic nitrogen (300 mg/l NH 4 NO 3 instead of 1650 mg/l), higher organic nitrogen (975 mg/l glutamine instead of 146 mg/l), high myoinositol (300 mg/l instead of 100 mg/l), 90 g/l maltose in place of 30 g/l sucrose, 2 mg/l PAA instead of IAA, phytagel in place of agarose, and the inclusion of 10 mg/l arabinogalactan-protein (AGP) (Sigma L 0650). Microspores cultured as drops on solidified medium with ovaries in 90 15 mm 2 Petri dish. Twenty ovaries, which were removed from freshly harvested spikes, or stored at 4 C for up to 1 wk, were used for co-culture 9. Plates were sealed with parafilm and incubated at 28 C in the dark for 30-32 d. Plant Regeneration After embryos reached the size of 2 mm, they were transferred to MMS5 7 differentiation medium for
COSKUN & SAVASKAN: PLANT REGENERATION THROUGH MICROSPORE CULTURE IN DURUM WHEAT 121 1-2 wks at 25 C with 16 h light period. The MMS5 medium was similar to MMS4 but contained lower maltose (30 g/l instead of 90 g/l), 0.2 mg/l PAA, 0.5 mg/l GA 3, 1 g/l casein, 690 mg/l proline and higher CuSO 4 (2.5 mg/l instead of 0.025 mg/l). When shoots were developed into an appropriate size, plantlets were transferred to MS medium with 30 g/l sucrose and omitted hormones and kept at 25 C with 16 h light. Statistical Analysis All the experiments were conducted in complete randomized designs (CRD) with at least three and more replications for each genotype. The frequency of divided microspores was calculated with the number that had divided by the d 10 in induction medium to the number of non-divided microspores by using a hemacytometer. Embryos were counted at d 30 in induction medium for all the experiments and categorized in two groups: large (>2 mm) and small (<2 mm, 0.5-2 mm). Only the large embryos were transferred to differentiation medium (MMS5). The numbers of divided microspores, embryo-like structures (ELS), embryo (small & large), green and albino plants and the total number of of plant regeneration were calculated for each pretreatment and genotype. Data were statistically analyzed using ANOVA with SPSS software program and means were compared with Duncan s Multiple Range Test (Duncan s Test). percentage of large (>2 mm) embryo formation. For pretreatments and interaction of cultivar PRT, all the parameters of microspore culture were also affected significantly. Effect of Pretreatments on Dividing Microspores and ELS It is clear from the studies that both mannitol (+4 C) and sorbitol (+4 C) pretreatments induced embryogenesis from isolated microspores of durum wheat. However, effect of mannitol (+4 C) was significantly improved in dividing microspores compared to sorbitol (+4 C). And also with mannitol (+4 C) pretreatment, microspores frequently formed a star-like or fibrillar structure, which is the deciding path that shows that microspores are switching to a sporophytic pathway (Fig. 1). The response difference between the pretreatments was also found significant (Table 3). The highest microspore dividing frequencies was observed with the pretreatment (+4 C) in all durum wheat genotypes and, as expected, it was the highest in bread wheat Gun-91 (64.28%). Among durum wheat genotypes, C-1252 showed the best result with a frequency of 56.25%. Earlier studies have shown that starvation and heat shock pretreatments induced the formation of embryos at high frequency in microspore cultures of Results and Discussion In four pretreatments (PRT) tested with five wheat cultivars, ANOVA showed that the number of divided microspores, embryo-like structures (ELS), embryos (small & large), green and albino plants, and the total of plant regeneration were strongly affected by cultivars and pretreatments (Table 2). Cultivars had a significant effect on dividing microspore percentage, while showed a lower influence on the Table 2 ANOVA of pretreatment effects on microspore culture parameters in wheat genotypes Source df F-value Dividing microspores Embryo-like structures (ELS) Fig. 1 Microspores with two different embryogenic stages ('star-like' structure) after 7 d of pretreatment. Embryo formation Small (<2 mm) Large (>2 mm) Green Plant regeneration Albino Cultivar 4 236.165** 32.376** 47.400** 8.917* 6.396** 25.476** 42.647** Pretreatment 4 30.382** 93.612** 51.412** 128.241** 29.982** 28.886** 38.834** (PRT) Cultivar PRT 16 116.385** 43.793** 33.095** 17.115** 29.500** 27.434** 26.970** Significant at **P<0.01, * P<0.05 Total
122 INDIAN J BIOTECHNOL, JANUARY 2017 Table 3 Mean effects of different pretreatments on isolated microspore culture response of wheat genotypes** Cultivar Pretreatments* Dividing microspores Embryo-like structures Embryo formation Small (<2 mm) Large (>2 mm) Green Plant regeneration Albino Kiziltan-91 I 27.70 ij 36.00 bc 19.00 gh 10.50 i 0.00 d 9.52 e-h 9.52 e-h II 43.75 d 32.54 d-g 25.00 bc 20.63 b 5.77 b 9.61 e-h 15.38 b III 37.50 e 30.45 gh 21.81 d-f 17.27 cd 0.00 d 7.89 h 7.89 h IV 16.60 o 21.66 kl 16.25 ij 10.42 i 0.00 d 8.00 gh 8.00 gh C-1252 I 31.25 gh 33.33 d-f 14.28 jk 12.86 g-i 0.00 d 3.70 i 3.70 i II 56.25 c 27.77 i 25.18 bc 17.77 c 4.16 c 10.42 d-g 14.58 bc III 33.33 fg 37.77 b 23.33 c-e 14.44 e-g 0.00 d 11.54 de 11.54 de IV 26.31 jk 33.85 c-e 18.23 hi 11.46 hi 0.00 d 9.09 e-h 9.09 e-h Mirzabey 2000 I 29.41 hi 31.46 e-h 13.79 k 10.77 i 0.00 d 4.00 i 4.00 i II 35.29 f 24.29 j 23.94 b-d 16.90 c-e 0.00 d 10.42 d-g 10.42 d-g III 31.25 gh 29.24 hi 21.23 e-g 15.09 d-g 0.00 d 9.37 e-h 9.37 e-h IV 22.22 mn 31.25 f-h 22.22 d-f 13.88 fg 0.00 d 5.00 i 5.00 i Total Kunduru-1149 I 23.53 lm 29.12 hi 15.38 jk 10.44 i 0.00 d 10.53 d-f 10.53 d-f II 29.41 hi 27.36 i 23.58 c-e 15.09 d-g 6.25 ab 12.50 cd 18.75 a III 25.00 kl 22.63 jk 20.00 f-h 14.74 e-g 0.00 d 10.71 d-f 10.71 d-f IV 21.05 n 20.00 l 15.00 jk 6.00 j 0.00 d 8.33 f-h 8.33 f-h Gun-91 I 60.00 b 41.00 a 34.00 a 15.50 c-f 4.00 c 20.00 a 19.35 a II 64.28 a 34.66 cd 35.11 a 25.33 a 7.02 a 14.03 bc 21.05 a III 41.66 d 32.86 d-g 26.19 b 15.24 d-g 0.00 d 15.62 b 15.62 b IV 38.48 e 32.22 d-g 23.88 b-d 13.33 f-h 0.00 d 12.50 cd 12.50 cd *Pretreatments: (i) Cold (+4 ), (ii) 0.4 M Mannitol (+4 C), (iii) 0.4 M Sorbitol (+4 C), & (iv) 0.4 M Mannitol (room temperature) **Means in a column followed by same letter are not significantly different at P<0.01 level by ANOVA test based on three replications wheat 10. Touraev et al 11 reported that using stress pretreatment on anthers at 33 C for several days produced embryos in microspore cultures with starlike structures and generated embryos following repeated symmetric divisions. In a cytological study comparing the effects of different pretreatments, mostly symmetric first divisions were observed during mannitol pretreatment, while only asymmetric first divisions were seen during cold pretreatment 4,12,13. In our study, we observed microspores representing a centralized nucleus surrounded by star-like cytoplasmic strands radiating towards cell wall with mannitol (+4 C) as well as with sorbitol (+4 C) pretreatments. The microspores had a vacuole fragmented by cytoplasmic strands with the nucleus positioned close to the microspore wall. When the two pretreatment applications were compared, mannitol treated microspores had the highest rate of conversion into embryos, while sorbitol treated microspores showed intermediate response. The frequency of ELS in different pretreatments varied 29.12-41.00% depending on the wheat genotypes (Table 3). Among the four pretreatments used in the study, cold (+4 C) pretreatment without polyols was more responsive to ELS and it was the highest in bread wheat Gun-91 (41.00%). Besides, sorbitol (+4 C) pretreatment resulted in the highest ELS frequency (37.77%) in durum wheat C-1252. Kiziltan-91 also yielded good results in cold (+4 C) pretreatment with a frequency of 36.00%. Mirzabey 2000 and Kunduru-1149 produced higher ELS in cold (+4 C) pretreatment. Pretreatment factors, temperature shock and starvation (mannitol) seemed to act in synergy in embryogenic induction 4. Using only temperature shock (+4 C) produced lower embryos and higher ELS.
COSKUN & SAVASKAN: PLANT REGENERATION THROUGH MICROSPORE CULTURE IN DURUM WHEAT 123 Effect of Pretreatments on Embryo Formation and Plant Regeneration Of four different pretreatments employed, mannitol (+4 C) significantly improved the formation of both large and small embryos in comparison to other pretreatments (Table 3). The number of small embryos produced in the pretreatment of mannitol (+4 C) was the highest in bread cultivar Gun-91 with a frequency of 35.11%. The formation of small embryo in Gun-91 was also higher in cold (+4 C) pretreatment. Among durum wheat genotypes, C-1252 and Kiziltan-91 yielded the best results for small embryos with a frequency of 25.18 and 25.00%, respectively. Regarding large embryos, Gun-91 produced the highest scores with a percentage of 25.33% in mannitol (+4 C) pretreatment. In durum wheat genotypes, Kiziltan-91 (Fig. 2) also had the second best response with a percentage of 20.63%. It is important to note that, besides mannitol (+4 C), sorbitol (+4 C) pretreatment also yielded a good performance compared to other pretreatments in all durum wheat genotypes. This study is the first report indicating the effects of sorbitol pretreatment on microspore embryogenesis in durum wheat cultivars. Earlier studies have also reported the microspore embryogenesis with mannitol pretreatment in bread wheat 14-16 and durum wheat 3. Several studies have pointed out that mannitol pretreatment gave the best results of microspore culture in cereals 7,17. Hu and Kasha 12 indicated that anther pretreatment with along with cold for 4 d substantially delayed the mitotic division of the nucleus, keeping all microspores at the same stage during pretreatment and also resulted in the formation of large numbers of embryos 3 in wheat. Our results showed that mannitol (+4 C) pretreatment had the highest microspore embryogenesis recovery potential across durum wheat cultivars. Moreover, the sorbitol (+4 C) pretreatment of microspores also yielded good results in embryogenesis of wheat. Overall, the highest regeneration of plantlets in microspore culture was observed in the pretreatment of mannitol (+4 C) with regard to all cultivars, both bread and durum wheat (Table 3). Gun-91 produced the highest green plant regeneration with a frequency of 7.02% as expected. Besides, Kunduru-1149 yielded the best results with a frequency of 6.25% among durum wheat genotypes (Fig. 3). However, no green plant regeneration was observed in genotype Mirzabey 2000 with all the pretreatments employed. In the pretreatment of mannitol (+4 C), the highest percentages of total plant regeneration frequency ranged 10.42-21.05% corresponding to Mirzabey 2000 and Gun-91, respectively. For the same pretreatment, in durum wheat genotypes, the highest total plant regeneration was observed in Kunduru-1149 with a frequency of 18.75%. Kiziltan-91 also yielded good results for green plant regeneration with high quality (Fig. 4). The highest regeneration Fig. 3 (a-b) Plant regeneration of durum wheat genotype Kunduru-1149: (a) Albino, & (b) Green plant Fig. 2 (a-d) Mature embryoids of different pretreatments in durum wheat genotype Kiziltan-91: (a) Cold pretreatment (+4 C), (b) 0.4 M Mannitol (+4 C), (c) 0.4 M Sorbitol (+4 C), & (d) 0.4 M Mannitol (room temperature). Fig. 4 (a-b) Plant regeneration of durum wheat genotype Kiziltan-91: (a) Albino, & (b) Green plant
124 INDIAN J BIOTECHNOL, JANUARY 2017 frequency of albino plants was observed in Gun-91 in all the pretreatments. It is also the only genotype where green plants were regenerated in cold (+4 C) pretreatment. Mannitol and sorbitol are isomers and have the same chemical formula and molecular weight, but differ in chemical properties and physical structure. Both mannitol and sorbitol can be used in further studies since their effects on albinism were similar with regard to the regeneration of plants. Moreover, owing to starch accumulation, it is likely that sorbitol is partly metabolized by microspores during pretreatment, which is consistent with its use as an alternative source of carbohydrates in culture media 18,19. In our study, sorbitol yielded the second best results after mannitol in dividing microspore count, embryo formation (>2 mm) and total plant regeneration. In the present study, it is evident from the results that genotype effect played an important role in the regeneration rate of green plants in microspore culture of durum wheat. Shariatpanahi et al 20 reported that the number of embryos in culture medium, total number of regenerated plants and frequency of green plants showed a significant difference between the genotypes. Liu et al 21 indicated that all tested genotypes responded to their method and formed green plants by microspore embryogenesis but genotypic differences were observed among applications with 2-HNA and stress pretreatments for regeneration to green plants. Of the four durum wheat cultivars tested in our study, Kunduru-1149 was the most responsive to the microspore culture. Despite having the lowest dividing microspore frequency, its regeneration capability was the highest among the four durum wheat tested with different pretreatments. Moieni and Sarrafi 22 reported that studies with bread wheat showed important genetic differences and relatively high heritability for androgenetic traits. Several studies have shown that the presence of the D genome facilitated a high androgenic response in bread wheat (AABBDD), while its absence was the cause for low induction of embryogenesis and green plant regeneration in durum wheat (AABB) 23,24. However, Zheng 4 observed a low heritability for albino plant regeneration, indicating that it may be possible to reduce albinos by controlling the culture conditions. Also in our study, Kunduru-1149 produced higher numbers of green plant regeneration with mannitol (+4 C) compared to other pretreatments. However, there was no green plant regeneration in all the genotypes with sorbitol (+4 C) and mannitol (room temperature) pretreatments. Improving an efficient microspore culture technique for all the wheat genotypes globally requires a good artificial manipulation of microspores and success in regeneration of large number of green plants. Due to genetic differences between cultures, it s very hard to apply the same method to all genotypes. The first critical factor in artificial manipulation involves treatment(s) that switche(s) microspores from naturally determined pollen formation to an alternative development leading to embryogenesis 4. The present study clearly shows that the pretreatment of mannitol (+4 C) was the best for improving microspore culture for green plant regeneration and Kunduru-1149 was the best cultivar in response to the culture technique as compared to the other Turkish durum wheat cultivars studied. Acknowledgement The present research was supported by The Scientific Research Projects of Suleyman Demirel University in Turkey (Project No: 1655-D-08). References 1 Ferrie A M R & Caswell K L, Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production, Plant Cell Tissue Organ Cult, 104 (2011) 301-309. 2 Cistue L, Romagosa I, Battle F & Echavarri B, Improvements in the production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture, Plant Cell Rep, 28 (2009) 727-735. 3 Labbani Z, De Buyser J & Picard E, Effect of mannitol pretreatment to improve green plant regeneration on isolated microspore culture in Triticum turgidum ssp. durum cv. Jennah Khetifa, Plant Breed, 126 (2007) 565-568. 4 Zheng M Y, Microspore culture in wheat (Triticum aestivum) Doubled haploid production via induced embryogenesis, Plant Cell Tissue Organ Cult, 73 (2003) 213-230. 5 Shim Y S, Kasha K J, Simion E & Letarte J, The relationship between induction of embryogenesis and chromosome doubling in microspore cultures, Protoplasma, 228 (2006) 79-86. 6 Cistue L, Soriano M, Castillo A M, Valles M P, Sanz J M et al, Production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture, Plant Cell Rep, 25 (2006) 257-264.
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