Prospects of androgenetic induction in Lupinus spp.

Transcription

1 Plant Cell Tiss Organ Cult (2008) 94: DOI /s ORIGINAL PAPER Prospects of androgenetic induction in Lupinus spp. Edyta Skrzypek Æ Ilona Czyczyło-Mysza Æ Izabela Marcińska Æ Maria Wędzony Received: 16 January 2008 / Accepted: 13 May 2008 / Published online: 24 May 2008 Ó Springer Science+Business Media B.V Abstract Anthers cultures of six Polish cultivars of pasture lupin (Lupinus L.) were examined for their androgenic response. Anthers with microspores at the uninucleate stage were isolated from flower buds and cultured in liquid media. Better viability of androgenetic structures was obtained when donor plants had grown under field as opposed to greenhouse conditions. A density of five anthers per 0.5 ml medium was more conducive to androgenetic induction than 25 anthers per 0.5 ml medium. Addition of 5% maltose to the induction medium and culture at 25 C without pre-treatment of flowers, buds or anthers promoted microspore release and division. The greatest frequency of androgenic callus, *70% was developed from cvs. Katon, Wat (white lupin), in contrast to cvs. Legat, Juno (yellow lupin), Polonez and Sonet (narrow-leafed lupin) with callus induction *30 40%. Despite various combinations of media tested, plant regeneration was not obtained from anther derived callus. E. Skrzypek I. Czyczyło-Mysza I. Marcińska M. Wędzony (&) Polish Academy of Sciences, The F. Górski Institute of Plant Physiology, Niezapominajek 21, Krakow, Poland niwedzon@cyf-kr.edu.pl M. Wędzony Pedagogical University of Cracow, Podchorą_zych 2, Krakow, Poland Keywords Androgenesis Anther culture Callus Lupinus albus L. angustifolius L. luteus Introduction Legumes are important both in the human diet as well as in animal nutrition in bio-organic farming systems. Due to their high protein and oil contents there is increasing interest in lupin breeding and cultivation. Lupins are promising crops for diverse weather conditions, for extreme or moderate climates and wide ranges of soil ph. Nevertheless, lupins like most legumes are recalcitrant in most tissue culture systems. Repeatable and efficient methods for plant regeneration have not been established. Androgenesis (microspore embryogenesis) is an important tool for production of doubled haploid (DH) lines (Małuszyński et al. 2003). Relatively poor progress has been achieved on androgenetic techniques in legumes (Croser et al. 2006). Several attempts have been made to develop anther and microspore culture systems for soybean (Glycine max L.) (Cardoso et al. 2004; Rodrigues et al. 2004; Lauxen et al. 2003; Kaltchuk-Santos et al. 1997; Ye et al. 1994), chickpea (Cicer arietinum L.) (Vessal et al. 2002; Huda et al. 2001; Bajaj and Gosal 1987; Khan and Ghosh 1983), alfalfa (Medicago sativa L.) (Zagorska and Dimitrov 1995; Tanner et al. 1990), pigeonpea (Cajanus cajan L.) (Kaur and Bhalla 1998), Phaseolus vulgaris (Muñoz-Florez and

2 132 Plant Cell Tiss Organ Cult (2008) 94: Baudoin 1994a, b; Peters et al. 1977). Few reports have been published on androgenesis induction in lupin (Lupinus L.) (Bayliss et al. 2004; Ormerod and Caligari 1994; Sator et al. 1983; Sator 1985). None of these efforts has resulted in the derivation of lupin DH; therefore these species have been defined as recalcitrant to androgenesis. First attempts in lupin androgenesis were described by Sator et al. (1983), who obtained only callus from anther culture of Lupinus polyphyllus. Plantlets regeneration was reported for anther culture of L. polyphyllus by Sator (1985), but chromosomes count suggested that they were diploid. Ormerod and Caligari (1994) observed embryo-like structures (ELS) with clearly defined cotyledons and radicals in liquid culture of Lupinus albus microspores. Recurrent somatic embryogenesis occurred following internal cleavage within the ELS or on the surface of the ELS. Direct conversion of ELS was not observed. It was difficult to recognize the origin of induced calluses and ELS, because anther cultures contained both haploid microspores and diploid tissues. Bayliss et al. (2004) reported a reproducible method of microspore culture that led to cell division and proembryo formation in Lupinus albus and L. angustifolius. Further development of these multicellular structures or pro-embryos was limited by the rigid outer exine layer. The purpose of the following research was to test previously described protocols and to modify methods to attempt to obtain Lupinus DH lines. We tested temperature pretreatments to buds and sugar compounds in the media. A new protocol is presented that repeatedly leads to androgenetic callus formation in anther culture using liquid media for L. albus, L. angustifolius and L. luteus. Material and methods Plant material We received seeds of Lupinus angustifolius (narrowleafed, cvs. Polonez and Sonet), L. albus (white, cvs. Katon and Wat) and L. luteus (yellow, cvs. Legat and Juno) from Breeding Company Poznańska Hodowla Roślin Sp. z o.o., Tulce, Poland; the seeds were coated with the antifungal agent Funaben T (Chemical Factory Organika Azot, Jaworzno, Poland) and sown either into open air experimental plots (May August) or in a greenhouse. In the latter case, four seeds were sown in each pot ( cm) filled with soil, peat and sand (3:2:1) and grown at 25/17 C for approximately 8 weeks before bud harvest. The soil in the pots was sprayed with a slurry of Rhizobium (Nitragina, prod. Biofood SC. ZPHU, Wałcz, Poland). Evaluation of microspore developmental stage After inflorescences were evaluated according to their length and developmental stage, anthers were isolated from flower buds, squashed and stained in a droplet of 4,6-diamidino-2-phenylindole (DAPI). Slides were stored in darkness at 4 C for 1 h and then analysed under a fluorescence microscope (Nikon Eclipse 600, UV light). On the basis of observation the appropriate bud stage for anther culture was established. Anther isolation and culture conditions Whole flower buds at various stages of development were harvested and either immediately used as a source of explants or stored in darkness at 4 C (for 2 or 5 days) or at 32 C (for 1 or 3 days) before isolation of anthers. Buds were surface-sterilized in 10% sodium hypochlorite for 15 min and rinsed five times with sterile water. Aseptically excised anthers were placed in 30 mm Petri dishes (5 or 25 anthers per dish) with 0.5 ml of liquid induction medium NNB5. The dishes were cultured in darkness at either 25 or 30 C. The NNB5 medium was composed of Nitsch and Nitsch macroelements (Nitsch and Nitsch 1969), B5 microelements (Gamborg et al. 1968), supplemented according to Ormerod and Caligari (1994) and adjusted to ph 5.8 prior to further processing. Depending on the experiment, the medium containing either 5% sucrose or 5% maltose was either autoclaved at 121 C for 25 min or filter sterilized (0.2 lm pores, 30 mm filters, Sigma Co.). A minimum of five Petri dishes per each combination of culture condition and cultivar was prepared. Induced calluses were transferred to any of four media: MS (Murashige and Skoog 1962), SH (Schenck and Hildebrandt 1972), B5 (Gamborg et al. 1968) and NNB5 as described above. All media were supplemented with 1.0 mg l -3 NAA and 1.0 mg l -3

3 Plant Cell Tiss Organ Cult (2008) 94: BA, solidified with 0.6% agar and cultured at 20 C under 16 h photoperiod (240 lmol(quantum) m -2 s -1 ). Statistical analysis The experiments were repeated thrice and data were analyzed by calculating mean ± standard error values and analysis of variance (ANOVA). Results Inflorescences and anthers were divided into three groups according to their developmental stage (Table 1, Fig. 1a, b). Buds of mm length, with the flower petals still inside the calyx and anthers circa 2 mm long were the most appropriate for anther culture since they contained microspores at the late uninucleate stage of development (Fig. 1c). Anthers isolated from plants grown in the greenhouse easily released microspores to the media; however androgenesis was not observed in these microspores and they died within 7 days after the start of the culture. Microspores released to medium from anthers of plants grown in the experimental field were more viable. Microscopic observations indicated that small microspores with dense cytoplasm did not divide. However, the larger microspores formed irregularly shaped callus within 2 3 months of culture (Fig. 1d). Figure 2 shows frequency of callus induction from anthers depending on temperature pretreatment of buds prior to anther isolation, kind of sugar in the induction medium and the anther density. The best callus induction was obtained from anthers isolated Table 1 Association of flower bud size with microspore developmental stage in lupin species Size and description of inflorecences Size and colour of anthers Microspore developmental stages [15 mm, light green, petals inside calyx 1 mm, light yellow Tetrads mm dark green, petals inside calyx 2 mm, yellow Late uninucleate [20 mm, dark green, top of petals outside calyx 3 mm, dark yellow Late uninucleate/early binucleate pollen Fig. 1 Lupin (Lupinus sp.) anther culture: (a) inflorescences of L. luteus cv. Juno, (b) anthers after isolation, (c) microspores at the late uninucleate stage (DAPI-stained), (d) 1-month-old callus with embryo-like structures (indicated by arrow) and (e) 2-month-old callus L. angustifolius cv. Polonez cultured on SH regeneration media

4 134 Plant Cell Tiss Organ Cult (2008) 94: Fig. 2 Induction of lupin callus depending on inflorescence pretreatment at 4 C (a) and 32 C (b), type of sugar (maltose or sucrose) in the induction medium and culture density (5 or 25 anthers per plate). (Bars represent means for L. angustifolius, L. albus and L. luteus with standard error, n = 30 Petri dishes per treatment) from freshly cut buds. The pretreatments with either 4 or 32 C did not improve microspore induction. Moreover, cold treatment lasting longer than 5 days caused death of microspores and 1 day of pretreatment at 32 C also almost completely destroyed microspore viability. The frequency of callus induction depended on anther density in the induction media. Higher induction occurred when five anthers were cultured per dish in comparison to 25 anthers (Fig. 2). Maltose stimulated callus induction more than sucrose. The temperature regime during the induction phase of anther culture influenced callus induction and the effect depended on the cultivar (Fig. 3). The most callus structures were induced at 25 C Lupinus angustifolius cvs. Polonez and Sonet. Anther viability and microspore androgenic development were reduced at 30 C for five of the six cultivars. L. luteus Fig. 3 The influence of temperature during lupin anther culture on callus induction. (Bars represent means with standard error, n = 15 Petri dishes per treatment) Fig. 4 The influence of autoclaved and filtered media on lupin callus induction. (Bars represent means with standard error, n = 15 Petri dishes per treatment) cvs. Legat and Juno and L. albus cv. Katon did not form callus at the higher temperature. Filtered medium significantly improved callus induction from microspores in comparison with autoclaved medium for four of six cultivars (Fig. 4). Only the narrow-leafed cultivars, Polonez and Sonet were exceptions. For subsequent experiments all media were filter sterilized. In medium with maltose, callus formation appeared to be greater than in medium with sucrose for narrow-leafed (L. angustifolius) and yellow (L. luteus) lupin (Fig. 5). White lupin (L. albus) showed a tendency to greater callus formation on medium with sucrose; however, none of the differences were statistically significant. The frequency of callus structures obtained depended on cultivar. Yellow lupin cvs. Legat and Juno showed the least callus induction and white lupin cvs. Katon and Wat the greatest.

5 Plant Cell Tiss Organ Cult (2008) 94: mg l -3 BA and 0.25 mg l -3 GA 3 (Fig. 1e). Attempts to regenerate plants failed. Discussion Fig. 5 The influence of sucrose (5%) and maltose (5%) on lupin callus induction for six cultivars. (Bars represent means with standard error, n = 5 Petri dishes per treatment) The callus structures induced in all experiments were transferred to MS, SH, B5 or NNB5 regeneration media with subculture to fresh medium every 4 weeks. Rapid growth of callus was initially observed on the all regeneration media, but most of the calluses became brown with necrotic spots within 1 month of culture (Fig. 6). Although white or darkgreen compact structures were formed on the surface of some calluses grown on SH medium (Fig. 1d, e) regeneration did not occur. Calluses obtained from cv. Polonez maintained viability over 6 months of culture on MS medium with 0.5 mg l -3 NAA, Fig. 6 Percent of green and necrotic brown calluses derived from three lupin cultivars on four regeneration media. (Bars represent means for L. angustifolius, L. albus and L. luteus with standard error, n = 5 Petri dishes per treatment) Since Sator et al. (1983) described the first efforts in lupin anther culture, a successful protocol for producing lupine doubled haploids has not been elaborated. This report describes callus induction from lupin microspores released to liquid medium from cultured anthers and further development of anther-derived callus on potential regeneration media, although regeneration was not achieved. Two years later Sator (1985) reported the regeneration of lupine plantlets from anther culture, however they were determined as diploid. In our experiments the greatest induction rate was found from microspores at the late uninucleate stage that was identified in *2 mm anthers excised from inflorescences of mm in length regardless of cultivar. Ormerod and Caligari (1994) reported that the binucleate stage of microspore development was highly responsive for androgenesis in lupin whereas Bayliss et al. (2004) found that microspores at the uninucleate stage produced many multicellular structures. For other species in which androgenesis has been reported, late uninucleate microspores or early bicellular pollen were optimal for androgenic induction (Custers et al. 2001; Datta 2001; Góralski et al. 1999). Various pretreatments of inflorescences or buds before anther or microspore isolation are frequently applied to raise androgenetic efficiency. The most frequently used are cold treatment, heat shock and/or sugar starvation (Bayliss et al. 2004; de Moraes et al. 2004; Kruczkowska et al. 2002; Indrianto et al. 2001; Smykal 2000; Kaltchuk-Santos 1997). Our experiment showed that heat pretreatment was inhibitory for lupin androgenesis and negatively influenced microspore viability. Low temperature pretreatment did not improve androgenic induction but slightly increased callus viability. According to Bayliss et al. (2004) optimal lupin microspore induction was achieved after storage of the buds for 3 days at 4 C, a finding that was not confirmed in our experiments. Similar to our results, Ormerod and Caligari (1994) reported androgenic induction in anther culture without any bud or inflorescence pretreatment.

6 136 Plant Cell Tiss Organ Cult (2008) 94: The stimulating effect of maltose on androgenesis has been described in isolated microspore culture of cereals (Indrianto et al. 2001; Puolimatka and Pauk 2000; Smykal 2000; Reynolds 1997; Scott et al. 1995) and in anther culture (Marciniak et al. 1998). Likewise, maltose compared to sucrose in the induction medium improved culture viability and androgenic response of lupin. The genotype and environmental conditions of donor plants play major roles in the androgenetic response (de Moraes et al. 2004; Custers et al. 2001; Datta 2001; Huda et al. 2001; Smykal 2000). In our experiments, field-grown plants provided a better source of flower buds compared to greenhouse grown plants. Among cultivars examined the greatest microspore induction occurred in white lupin cvs. Wat and Katon and narrow-leafed lupin Polonez. Anther derived calluses of lupin did not regenerate any plants even though they were maintained for many months and formed green meristemoids or white structures resembling embryos. The failure of these structures to convert into plants may have been due to nonoptimised regeneration medium with insufficient or improper growth regulators or too low/too high carbohydrate level. Bayliss et al. (2004) reported that further development of embryo-like structures was limited by a strong exine layer surrounding dividing microspores, which is certainly not the case in our experiments, as the structures in our experiments had released from the pollen wall. Future research on improvements of protocol for lupin should concentrate on optimisation of media and regeneration conditions. Acknowledgement This study was supported by COST /E 189/SPB/COST/P-06/DZ 585/ References Bajaj YPS, Gosal SS (1987) Pollen embryogenesis and chromosomal variation in cultured anthers of chickpea. 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