Agricultural and Food Science in Finland 231 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 9 (2000): 231–238. © Agricultural and Food Science in Finland Manuscript received February 2000 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 9 (2000): 231–238. Maintenance of male sterile germplasm in Brassica rapa by in vitro propagation Yang-Dong Guo Laboratory of Plant Physiology and Molecular Biology, Department of Biology, FIN-20014 University of Turku, Finland, e-mail: yaguo@utu.fi Tarja Niemelä, Unto Tulisalo Mildola Ltd, Anttila Research Farm, Anttilantie 30, FIN-04300 Tuusula, Finland Seppo Pulli Laboratory of Plant Physiology and Molecular Biology, Department of Biology, FIN-20014 University of Turku, Finland An efficient tissue culture system for plant regeneration, from mature cut branches, was established to maintain male sterile material in Brassica rapa L. The new-growth immature pods from the cut branches were used as explants; they gave better results in callus initiation (37 calli from 25 ex- plants) and shoot formation (17 shoots from 75 explants) than flower buds and branch stems. Auxin [2,4-dichlorophenoxyacetic acid (2,4-D), 2 to 5 mg l-1] and cytokinin [6-benzylaminopurine (BA), 2 to 4 mg l-1] were essential in callus and shoot formation, respectively. Callus initiation and shoot regeneration capacities were genotype dependent. The regenerated plants were male sterile and were used in breeding programs. Key words: Brassica rapa L., callus, in vitro micropropagation, plant growth regulators, shoots Introduction Plant regeneration from somatic cells is possi- ble either via shoot/root organogenesis or via so- matic embryogenesis. Both pathways have re- sulted in regenerants from numerous species (Lörz et al. 1988). One of the most useful appli- cations of plant tissue culture lies in the possi- bility of cloning a particular genotype. This can be especially important if the desired plant is a hybrid or an allogamous species, due to the fact that the sexually-derived progeny of these types are no longer identical to the original genotype (Linacero and Vazquez 1990). Clonal propaga- tion of plants is based on the concept of totipo- tency. It is a prerequisite for most applied uses of plant tissue cultures. Effective plant tissue culture techniques have been developed for sev- eral of the economically important species of mailto:yaguo@utu.fi 232 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Guo, Y.-D. et al. Maintenance of male sterile material in Brassica rapa Brassica from explants of leaves, hypocotyls, petioles, stem layers and protoplasts (Bhattach- arya and Sen 1980, Li and Kohlenbach 1982, Eapen et al. 1989, Kirti and Chopra 1990). Turnip rape (Brassica rapa L.) is the most important oil crop in Finland. Cytoplasmic male sterility was used in turnip rape breeding work. This cytoplasmic male sterility (cms), original- ly found by Ogura (1968), was transferred to B. oleracea and B. napus. Male sterile B. napus cybrids were then produced through protoplast fusion (Pelletier et al. 1983) to generate male sterile lines with highly stable male sterility, improved nectar secretion and a high productiv- ity (Pelletier et al. 1987). This OGU-INRA cy- toplasmic male sterility was transferred to Brassica rapa by back-crossing. The in vitro propagation method was used to save the male sterile breeding material in this study. This meth- od facilitated recycling of the same sterile A-line (cms) genome and the crossing of it with certain R-lines (fertility restoring) in descending gen- erations in order to study the maternal impact on F1 hybrids. Brassica rapa is more recalcitrant in tissue culture than other Brassica species. In this study, cut branches were collected, cold treated and forced to induce new buds. Old stems and new- growth tissues such as flower buds and new- growth immature pods were then used as ex- plants for in vitro propagation. The best results were achieved from new-growth immature pods of the harvested male sterile plants in callus in- itiation and shoot regeneration tests. Complete plants were regenerated. Male sterile breeding material was thus maintained via in vitro propa- gation using immature pods as explants. Material and methods Cut branches from 121 different male sterile breeding line plants, under cross-testing in the greenhouse, were used as explant sources. The greenhouse growth condition for donor plants was 20°C/17°C day/night temperature and a 16- h photoperiod at 150 µmol m-2 s-1 supplemented by fluorescent lamps. The breeding lines includ- ed 91451-A, 92549-A, 4001-A, 4002-A and 4003-A, all Mildola’s female breeding lines, from Brassica rapa hybrid program. Parkland, an old Canadian variety, was used as a control in this program. All test lines, except for the con- trol, have Ogura radish (OGU-INRA) cytoplas- mic male sterility. Vegetative cut branches were stored with stalks in water in a 4°C cold room with 8-h pho- toperiod at 20 µmol m-2 s-1 for one month, then, these cut branches were transferred to a green- house to induce new growth. The growth condi- tion for inducing flower buds and immature pods was 25°C and a 16-h photoperiod at 150 µmol m-2 s-1 supplemented by fluorescent lamps. The branch stalks were in tap water and water changed every three days. Branch stems, flower buds and new-growth immature pods of the harvested branches were surface sterilized in 70 (v/v) ethanol for 1 min, followed by 5 min in a 10% sodium hypochlo- rite solution containing one drop of Tween-20 and then rinsed three times with sterile distilled water. The following explants were used to ini- tiate cultures on semi-solid medium: the suita- ble size of flower bud was between 3 and 6 mm; branch stem explants were 3 mm thick transverse sections; 5–10 mm long immature pods contain- ing embryos, were cut into 4 to 5 mm explants. The explants were plated on semi-solid cal- lus induction media. The culture medium con- sisted of B 5 (Gamborg et al. 1968) macro- and micro-nutrients, inositol 100 mg l-1, nicotinic acid 1 mg l-1, pyridoxine-HCl 1 mg l-1, thiamine- HCl 5 mg l-1, L-glutamine 2 mg l-1, L-alanine 10 mg l-1, casein hydrolysate 200 mg l-1, glycine 2 mg l-1, sucrose (3%, w/v) and PhytagelTM (0.3%, w/v). All media used in these experiments were sterilized by autoclaving at 1.1 kg/cm2 and 120°C for 20 min. The explants were cultured in 9 cm Petri dishes containing 35 ml of sterile medium in darkness at 27°C. The effect of growth regu- lators on callus induction was investigated by culturing explants on induction media supple- 233 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 9 (2000): 231–238. mented with different concentrations of auxin [2,4-dichlorophenoxyacetic acid (2,4-D), 1–6 mg l-1] combined with 0.1 mg l-1 kinetin (6-furfur- ylaminopurine). Each treatment consisted of three replicates each with 25 explants. After a four week induction period, the calli were subcultured onto the same medium for three to four weeks. Calli between 5 and 10 mm in size, were transferred to shoot regeneration me- dium consisted of B 5 basal components contain- ing 6-benzylaminopurine (BA) (1–5 mg l-1) in combination with one of three low levels of aux- in, naphthaleneacetic acid (NAA) 0.2 mg l-1, 3- indoleacetic acid (IAA) 0.2 mg l-1 or 2,4-D 0.1 mg l-1. Shoot regeneration was maintained at a light intensity of 50 µmol m-2 s-1 and a 16-h pho- toperiod supplemented by fluorescent lamps and a temperature of 25°C. For rooting, regenerated shoots were transferred to half-strength, hor- mone-free B 5 medium at conditions same as that for shoot regeneration. The rooted plantlets were transferred into 15 cm plastic pots containing peat/sand mixture to a greenhouse under control- led conditions employing 25°C/18°C, 16 h pho- toperiod. The light intensity was about 150 µmolm-2 s-1 supplemented by fluorescent lamps. Plantlets were covered with glass beakers dur- ing the first week of acclimatization to prevent desiccation of plants. Results and discussion Explant sources There were differences in callus induction and shoot propagation capacities among the three sources of explants (Table 1). The callus initiat- ed from new-growth immature pods was solid, compact and grew rapidly (Fig. 1a). The callus from flower buds grew slowly, and was loose. The callus from branch stems grew slowly, turned brown in colour and died after several subcultures. Green shoots could not be obtained from branch stem derived callus. Callus induc- tion and shoot formation was much better from immature pod explants than from flower bud and stem explants. The explant source acts an important role in micropropagation of Brassica species. In Brassi- ca rapa ssp. Pekinensis, the successful explant for micropropagation was the cotyledon (Kuo and Tsay 1977), it represented a significant im- provement in regeneration of Chinese cabbage in comparison with axillary buds that were cul- tured. Young hypocotyl explants were used for somatic embryogenesis and plant regeneration in six Brassica species (Dietert et al. 1982, Yang et al. 1991, Narasimhulu et al. 1992). Hypocotyl segments, stem and leaf explants from seedlings were used in somatic embryogenesis in Brassi- ca nigra (Narasimhulu et al. 1992). In Brassica napus, in vitro plant formation was induced from internodal segments (Kartha et al. 1974), coty- ledon, hypocotyls and stem sections (Khehra and Mathias 1992), axillary buds (Polowick and Sawhney 1991) and thin cell layer explants (Kli- maszewska and Keller 1985, Shu and Loh 1991). In the present study, branch stems, flower buds and immature pods were used as explants in mi- cropropagation due to old and wilting donor materials. The immature pods gave the best re- sults for callus induction and plant regeneration. The age of the donor plant (explant) is a crit- Table 1. The number of calli formed and shoots regener- ated in turnip rape male sterile breeding line (4003A-26). The modified B 5 medium containing 5 mg l-1 2,4-D and 0.1 mg l-1 kinetin was used in callus induction. In shoot regen- eration, B 5 medium with 2 mg l-1 BA and 0.1 mg l-1 2,4-D was employed. Data is the average of three replicates, 25 explants per replicate. Explant source No. callus + SD* No. shoot (total) Immature pod 37 + 9 17 Flower bud 16 + 2 5 Branch stem 7 + 3 0 * SD = standard deviation. 2,4-D = 2,4-dichlorophenoxyacetic acid Kinetin = 6-furfurylaminopurine BA = 6-benzylaminopurine 234 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Guo, Y.-D. et al. Maintenance of male sterile material in Brassica rapa ical factor affecting callus initiation and shoot regeneration in Brassica species (Yang et al. 1991). Generally, young tissue is used as an op- timal explant, the old explants are always avoid- ed in plant tissue culture. In the present study, 121 male sterile breed- ing line plants were used as explant sources. These donor plants were under cross-testing in the greenhouse at the time that the branches were cut. Only a few breeding lines were selected for micropropagation as determined by their agro- nomic characteristics and crossing ability in de- scending generations in order to study the ma- ternal impact on F1 hybrids. Therefore, the cut branches were stored (for one month at 4°C) awaiting greenhouse test results. Using this method, time and labour could be saved. How- ever, these cut branches were old and conven- tional micropropagation techniques could not be applied. After branches were moved to the greenhouse at 25°C, the new-growth immature pods and new-growth flower buds became usable. These new-growth organs with active metabolism Fig. 1. Callus initiation and shoot regeneration in Brassica rapa male sterile material (4003A-6) using immature pods. The modified B 5 medium containing 4 mg l-1 2,4-D and 0.1 mg l-1 kinetin was used in callus induction from immature pod. In shoot regeneration, B 5 medium with 2 mg l-1 BA and 2,4-D 0.1 mg l-1 was employed. Abbreviations: 2,4-D = 2,4-dichloroph- enoxyacetic acid, Kinetin = 6-furfurylaminopurine, BA = 6-benzylaminopurine. (a) Callus initiation from an immature pod explant after two weeks of culture (8 x). (b) Shoot formation from callus in regeneration medium (8 x). (c) Well developed plantlet from immature pod propagation. (d) A flower from regenerated plant with male sterile character. 235 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 9 (2000): 231–238. yielded calli and shoots with a relatively high regularity. Influence of 2,4-D concentration on callus initiation B 5 basal medium (Gamborg et al. 1968) was used as callus induction and shoot regeneration me- dium in this study. Auxin and cytokinin are es- sential in callus induction and shoot formation, respectively. In this study, 2,4-D was used as the auxin at 1 mg l-1, 2 mg l-1, 3 mg l-1, 4 mg l-1, 5 mg l-1, 6 mg l-1 for callus induction, combined with a low level of cytokinin (kinetin 0.1 mg l-1). Con- centrations of 2,4-D between 2 and 5 mg l-1 gave the best result for callus induction. However, there was a genotype difference. For clone 92549A-8, 4–5 mg l-1 2,4-D promoted the callus initiation effectively whereas for clones 4003A- 26 and 4002A-5, the best dosages of 2,4-D were 5 mg l-1 and 4 mg l-1, respectively (Fig. 2). The importance of 2,4-D in callus initiation and growth in Brassica species has been recognized (Bajaj and Nietsch 1975, Dietert et al. 1982, Shu and Loh 1991). Dietert et al. (1982) reported that a high level of 2,4-D may inhibit callus growth, but the presence of low levels of this auxin is required for callus proliferation and for inhibi- tion of root development in certain cultivars of B. oleracea and B. napus. Influence of BA on shoot formation The function of BA in Brassica species micro- propagation has been reported. Deng et al. (1991) described that in Chinese cabbage (B. rapa ssp. Pekinensis), the concentration of BA which pro- moted the highest rate of shoot induction showed clonal variation and was in the range of 44.4 to 177.6 µM. In B. napus, BA alone (5 µM) induced multiple shoot formation on stem explants (Kartha et al. 1974). In this work, the combina- tions of auxin (NAA 0.2 mg l-1, IAA 0.2 mg l-1 and 2,4-D 0.1 mg l-1) and cytokinin (BA 1–5 mg l-1) were investigated. In a shoot regeneration ex- periment using clone 4003A-8, and in the pres- ence of NAA 0.2 mg l-1, IAA 0.2 mg l-1 or 2,4-D 0.1 mg l -1, the optimal BA concentrations were 3 mg l-1, 4 mg l-1 and 2 mg l-1, respectively (Fig. 3). Shoots could be obtained after 4 to 6 weeks when calli were cultured on regeneration medi- um (Fig. 1b). When the shoots were 1 cm high, they were cut from callus and subcultured to the Fig. 2. Effect of auxin (2,4-D = dichlorophenoxyacetic acid) con- centration combined with 0.1 mg l-1 kinetin (6-furfurylaminopurine) on callus initiation from immature pod of turnip rape in vitro propa- gation. Four replicates, 25 ex- plants per replicate. Vertical bars indicate the standard error of the mean. 236 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Guo, Y.-D. et al. Maintenance of male sterile material in Brassica rapa continued from preceding column Breeding lines No. callus/dish Shoot formation (total) – 7 33 + 5 7 –10 19 + 3 5 –14 23 + 3 8 –15 18 + 1 28 –17 20 + 4 9 –18 31 + 3 0 –20 28 + 6 0 –22 27 + 9 5 4003A – 1 25 + 3 0 – 5 23 + 9 6 – 6 31 + 8 17 – 7 17 + 4 0 – 8 25 + 9 29 –10 25 + 5 4 –14 24 + 1 15 –16 13 + 3 0 –17 35 + 3 3 –18 23 + 3 1 –19 26 + 2 0 –20 15 + 3 10 –21 31 + 4 3 –22 23 + 6 14 –25 34 + 3 0 –26 35 + 2 8 * SD = standard deviation. 2,4–D = 2,4–dichlorophenoxyacetic acid Kinetin = 6–furfurylaminopurine BA = 6–benzylaminopurine Table 2. Genotype effect on callus and shoot formation in B. rapa male sterile breeding materials propagation. Im- mature pods were used as explants in callus induction ex- periments. The modified B 5 medium containing 4 mg l–1 2,4–D and 0.1 mg l–1 kinetin was used in callus induction. In shoot regeneration, B 5 medium with 2 mg l–1 BA and 0.1 mg l–1 2,4–D was employed. Means + standard deviation are from three replicates, 25 explants per replicate. Breeding lines No. callus/dish Shoot formation (total) 91451A – 1 31 + 3* 5 – 4 25 + 3 11 – 5 17 + 3 0 – 6 34 + 4 0 –11 20 + 4 1 –12 15 + 5 25 –14 16 + 4 12 –16 24 + 4 3 –19 24 + 6 0 –20 26 + 8 7 –21 24 + 2 10 –23 39 + 1 6 –24 24 + 5 0 –25 9 + 3 5 –28 21 + 6 0 92549A – 5 17 + 4 10 – 6 15 + 3 0 – 8 33 + 3 7 – 9 25 + 2 3 –11 37 + 3 2 –12 20 + 6 11 –14 18 + 1 15 –15 25 + 9 0 Parkland – 3 23 + 5 4 – 4 7 + 3 0 – 5 14 + 4 0 – 6 30 + 3 5 – 7 25 + 5 11 – 8 33 + 4 4 – 9 24 + 6 17 –10 28 + 7 5 –13 24 + 3 0 –14 20 + 3 13 –15 33 + 4 7 –16 34 + 6 9 4001A – 1 25 + 7 5 – 3 24 + 4 15 4002A – 2 35 + 3 5 – 3 31 + 8 7 – 4 27 + 8 0 – 5 25 + 4 24 – 6 37 + 2 7 continued on next column same regeneration medium. The shoots were transferred to a culture jar containing half- strength, hormone-free B 5 medium for rooting when they were over 2.5 cm high (Fig. 1c). The regenerated plants grew normally and had male sterile character (Fig. 1d). All the clones showed a high degree of phenotypic uniformity within the clone. Genotype effect on callus and shoot formation Among 121 branches, callus was initiated from 66 and shoots from 48 branches which indicates a genotype difference in propagation ability of 237 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 9 (2000): 231–238. Fig. 3. Effect of hormone combi- nations on shoot formation in B. rapa (4003A – 8) in vitro propa- gation. Means are from four rep- licates, 25 explants per replicate. Vertical bars indicate the standard error of the mean. B. rapa (Table 2). The callus initiation and shoot regeneration capacities were genotype depend- ent in this study. Acknowledgments. We thank Mr. Jukka Karhu for techni- cal assistance. This work was supported by Mildola oy and the Finnish Ministry of Agriculture and Forestry. References Bajaj, Y.P.S. & Nietsch, P. 1975. In vitro propagation of Red Cabbage (Brassica oleracea L. var. capitata). 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SELOSTUS Isästeriilin kevätrypsin geeniaineksen ylläpito in vitro mikroviljelyllä Yang-Dong Guo, Tarja Niemelä, Unto Tulisalo ja Seppo Pulli Turun yliopisto ja Mildola Oy Rypsin (Brassica rapa) solukkoviljely on ristikuk- kaisten kasvien solukkoviljelyistä vaikeinta. Tämän tutkimuksen tarkoituksena oli pelastaa in vitro mik- roviljelyllä valinnassa olevat OGU-INRA isästeriilit CMS-kevätrypsit takaisin valintakiertoon. Tutkimuk- sessa yritettiin in vitro mikroviljellä tuleentuneen sa- don tuottaneiden ja kylmäkäsittelyn saaneiden isäste- riilien kasvien lehdistä ja varsista uusia, vihreitä kas- veja. Tehtävä epäonnistui, koska tutkittava materiaali ei kyennyt uusiutumaan ikänsä ja mikrobi- ja sieni- saastuntansa vuoksi. Jatkotutkimuksessa sama, lähes eloton, tutkimusmateriaali siirrettiin kasvihuoneen optimiolosuhteisiin ja nesteravintoviljelyyn. Kasvit pakotettiin kukintaan, joista edelleen epäkypsät lidut saatiin kasvattamaan kallusta, joka voitiin regeneroi- da isästeriiliksi lähtömateriaaliksi. Sekä kalluksen kehittyminen että regenerointi todettiin genotyyppi- riippuvaisiksi. Kehitetty menetelmä mahdollistaa ar- vokkaan CMS-materiaalin uudelleenkäytön kypsää satoa vaativan valintaprosessin jälkeen. Ennen kaik- kea menetelmä mahdollistaa CMS-äidin uudelleen ja uudelleen testaamisen useiden tekijöiden suhteen, mikä muutoin ristikukkaisella kasvilla olisi mahdo- tonta. Title Introduction Material and methods Results and discussion References SELOSTUS