Turkish Journal of Biology http://journals.tubitak.gov.tr/biology/
Research Article
Turk J Biol (2014) 38: 593-600 © TÜBİTAK doi:10.3906/biy-1403-29
Effect of polyamines on in vitro anther cultures of carrot (Daucus carota L.). Krystyna GÓRECKA*, Waldemar KISZCZAK, Dorota KRZYŻANOWSKA, Urszula KOWALSKA, Agata KAPUŚCIŃSKA Research Institute of Horticulture, Skierniewice, Poland Received: 12.03.2014
Accepted: 13.05.2014
Published Online: 05.09.2014
Printed: 30.09.2014
Abstract: This study examines the influence of the polyamines putrescine and spermidine on the efficiency of androgenesis in anther cultures of 2 carrot cultivars, Kazan F1 and Narbonne F1, and the effect of putrescine on the process of plant regeneration from androgenetic embryos. In the Kazan F1 variety, an increase in the number of obtained embryos was achieved using each of the 2 polyamines separately and in combination. In contrast, no beneficial effects of polyamines on the efficiency of androgenesis were observed in the Narbonne F1 variety. Putrescine added to the regeneration medium increased the number of obtained plants. For embryos obtained on induction medium without putrescine, the best concentration was 160 mg putrescine in 1 L of regeneration medium, and for those obtained on the induction medium with putrescine the best concentration was 0.5 mg putrescine per 1 L. All of the resulting plants, both in the experimental and control combinations, had a doubled set of chromosomes. The vast majority of them were homozygous for both isoenzymes, glucose-6-phosphate isomerase and aspartate aminotransferase, and the distribution of homo- and heterozygous received combinations with the polyamines and in the control was very similar. Key words: Carrot, androgenesis, putrescine, spermidine, regeneration
1. Introduction The carrot is an economically important vegetable whose importance increases through broadening the scope of its potential uses. Carrot juice, or mixed juices in which carrot is one of the main ingredients, are gaining popularity along with fresh, “one-day” juices with no preservatives. The carrot root is also a component of many frozen foods. In addition to its food uses, the pharmaceutical industry is also interested in the carrot as a source of carotene. To meet the demands of producers and consumers, intensive breeding work on the carrot continues. The carrot market, like that of other vegetables, is dominated by hybrid varieties. The breeding of such cultivars by traditional methods is difficult and time-consuming; however, the application and use of in vitro androgenesis helps to reduce the homozygotation period to a few months. Until recently, the carrot was considered a difficult species as far as androgenesis is concerned. At the Research Institute of Horticulture (formerly the Research Institute of Vegetable Crops) in Skierniewice, Poland, a method of obtaining homozygous carrot plants has been developed. Currently, it is still being refined (Górecka et al., 2005a, 2005b, 2009). The efficiency of embryogenesis in anther cultures is influenced by many factors (Bajaj, 1990; Savaşkan et al., 1999; Özkum Çiner and Tıpırdamaz, 2002; Datta, 2005; Bhojwani and Dantu, 2010). One of the most important * Correspondence:
[email protected]
is the induction medium and its various components. Polyamines are components of the induction medium that are among the factors that may help improve the efficiency of embryogenesis in anther cultures remove (Dewi et al., 2004; Chiancone et al., 2006; Hema and Murthy, 2008). Polyamines are organic ammonia derivatives in which 1, 2, or 3 hydrogen atoms are replaced by alkyl or aryl groups. They are present in all living organisms. In plants, they are located mainly in the cell wall, vacuoles, chloroplasts, and mitochondria and within the nucleus and nucleolus (Niklas et al., 1998). Their existence has been known for over 100 years, but their role in life processes has been recognized relatively recently. Currently, some scholars consider them to be a new group of plant growth regulators. However, the concentrations at which they work are much higher when compared to typical growth substances (Galston and Kaur-Sawhney, 1995). Polyamines are involved in many vital plant functions (Sińska, 1986); for example, they regulate the growth and development of plant organisms and organogenesis, take part in the initiation of buds and differentiation of flower buds, and aid in fruit formation and the growth of roots. They also affect pollen viability. Polyamines play a significant role in many important physiological and metabolic processes, including, among others, the phosphorylation of proteins and transformation
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GÓRECKA et al. / Turk J Biol of phosphatidylinositols. They also prevent the degradation of cytoplasmic membranes and excessive concentrations of ethylene, thus delaying the ageing process (Tiburcio et al., 1993). They are involved in plant responses to stresses caused by different abiotic factors (Cohen, 1998; Górecka et al., 2007; Szafrańska et al., 2011). They play a role in maintaining the balance between cations and anions in cases of mineral deficiency (Watson and Malmberg, 1996). They have a stabilizing effect on the nuclear chromatin, lipoprotein membranes, and the cytoskeleton, and also take part in the activation of cell divisions. By participating in the crosslinking of cytosolic proteins, polyamines stabilize the cell cytoskeleton. If there are no polyamines present, cells are not capable of replication, and the actin filament bundles and microtubules in the cytoskeleton cease to exist (Jabłońska-Trypuć and Czerpak, 2007). Exogenous polyamines improve regeneration in tissue cultures (Kakkar et al., 2000; Paul et al., 2009). There is a large body of literature concerning the role of polyamines in the process of somatic embryogenesis (Kumar et al., 1997; Liu et al., 1997). Much of it points out the importance of exogenous polyamines in improving the process of somatic embryogenesis (Kevers et al., 2000; Takeda et al., 2002; Silveira et al., 2006; Steiner et al., 2007; Paul et al., 2009). There are relatively fewer reports on the participation of polyamines in gametic embryogenesis (Forodi et al., 2009). An improvement in the efficiency of gynogenesis in onion has been achieved through the exogenous application of polyamines (Martinez et al., 2000; Geoffriau et al., 2006). The role of polyamines in androgenesis has been investigated by Tiainen (1992) in potatoes, Dewi et al. (2004) and Dewi and Purwoko (2008) in rice, Kumar et al. (2004) in cucumbers, Hema et al. (2008) in niger (Guizotia abisinica L.), and Chiancone et al. (2006) in clementines. The aim of the experiments described here was to investigate the effect of the 2 most frequently used polyamines, putrescine and spermidine, on the efficiency of androgenesis in carrots and the effect of putrescine on the regeneration of plants from androgenetic embryos. 2. Materials and methods The experiments were conducted with 2 carrot cultivars: Kazan F1 and Narbonne F1. After vernalization in cold storage at 4 °C, carrot roots were planted in a peat-sand (1:3 v/v) substrate, enriched with compound fertilizer Azofoska at 1.2 kg m–3 of the used peat, with the addition of chalk in an amount of 8 kg m–3 of the substrate. Donor plants were grown in a growth chamber under a controlled temperature of 20 °C during the day and 16 °C at night. The light intensity was 30 µmol m–2 s–1. Care and protection of the plants was carried out according to the recommendations for carrots grown for flowering.
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The anther cultures were carried out according to the procedure developed and described by Górecka et al. (2005a, 2005b). Plants with heterozygous lanes of bands for both tested isoenzymes – glucose-6-phosphate isomerase (GPI; EC 5.3.1.9) and aspartate aminotransferase (AAT; EC 2.6.1.1) – were used as donor plants. The stage of microspore development was determined by preparing crushed specimens of anthers stained with 1 drop of acetocarmine, which were examined under a microscope with immersion at a magnification of 100×. Based on the results of our previous studies (Kiszczak et al., 2005), we selected for the experiments flower buds containing predominantly microspores at the uninuclear stage. Donor plants were grown so that each would produce a few well-developed inflorescences, which means that either the plant was left with the main shoot and a few shoots of the first order, each terminated with one inflorescence, or that the plant had a few shoots of the same order, each with a single inflorescence. B5 medium (Gamborg et al., 1968) containing 100 mg dm–3 L‑serine, 500 mg L‑glutamine dm–3, naphthalene acetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.1 mg dm–3 each and 100 g dm–3 sucrose was used. To study the effect of polyamines on the efficiency of androgenesis, we chose putrescine and spermidine, the 2 most studied and described in the literature. The experimental combinations were as follows: 1. B5 control medium, 2. B5 + 322 mg dm–3 putrescine, 3. B5 + 312 mg dm–3 spermidine, 4. B5 + 322 mg dm–3 putrescine + 312 mg dm–3 spermidine. Polyamine concentrations were chosen based on other studies about androgenesis, particularly that of Chiancone et al. (2006). The flasks with anthers were placed in the dark at 27 °C. Starting from 3 weeks after the establishment of the cultures, we began observations of embryo formation. The flasks containing embryos were exposed to continuous light while maintaining the temperature of 27 °C. The efficiency of androgenesis was expressed as the number of embryos per 100 laid out anthers. When the embryos had increased in size and were beginning to turn green, they were transferred to the regeneration medium. The regeneration of plants from androgenetic embryos was carried out according to the procedure described and patented by our team in the Polish Patent Office (Patent No. 208426). The medium used was B5 without hormones and amino acids, with the sucrose content reduced to 20 g dm–3. Embryos were placed singly in test tubes in a growth room at 20 °C; light intensity was 30 µmol m2 s–1 and the
GÓRECKA et al. / Turk J Biol day lasted 16 h. Passage was performed several times onto the same medium. Nonrooted rosettes, incipient rosettes, and secondary embryos (Figure 1A) were transferred onto a fresh medium of the same composition. Complete plants with a well-developed root (Figure 1B) were planted after removal from glass into multiple pots with a sand-peat (3:1 v/v) substrate enriched with 1g dm–3 Universol Yellow (NPK 12:30:12 + 2MgO + microelements) fertilizer and were placed on a capillary mat in a small plastic tent to ensure 100% humidity (Figure 1C). The tent was placed in a growth room at a temperature of 20 °C during the day and 16 °C during the night, with a 16-h day. After approximately 3 weeks, ventilation was started (Figure 1D). After hardening, plants were transplanted into larger containers with a substrate. An experiment was also carried out on the effect of putrescine on the regeneration of plants from androgenetic embryos. The following media were used: without putrescine, and with 0.5 mg, 160 mg, and 322 mg
A
putrescine per 1 L. We decided to use these putrescine concentrations because various authors have described their positive effects on regeneration (Kakkar et al., 2000; Dewi et al., 2006; Paul et al., 2009). Embryos obtained on the control induction medium and the induction medium with putrescine were laid out onto these regeneration media. Adapted plants were assessed, and ploidy was determined first. In our previous studies we obtained cytological test results by counting the chromosomes that corresponded to the results received through cytometric analyses involving determination of the amount of nuclear DNA by means of a flow cytometer. The tests were conducted according to the procedure of Partec, with modifications. Because of high levels of phenols in the plant material, which exhibit inhibitory activity towards DNA staining by fluorochrome, it was impossible to use the standard buffer, so 0.1% polyvinylpyrrolidone (PVP-40) was used instead. The material for the tests were young leaves. Approximately
A
C
B
D
Figure 1. A: Plant material received from androgenetic embryos in regeneration; B: plants obtained in regeneration process; C: plant adaptation in plastic tunnel; D: plants after adaptation.
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GÓRECKA et al. / Turk J Biol 20 mg of leaf tissue was chopped up with a razor blade in a petri dish with 2 mL of CyStain DNA lysis buffer and PVP-40. Following the addition of fluorochrome 4’,6-diamidino-2-phenylindole (DAPI) in the amount of 0.1 mg in 1 mL, the samples were filtered through a nylon filter with 50-µm pores (Partec Cell Trics, Partec GmbH, Münster, Germany). The prepared samples were analyzed by means of a CAII flow cytometer (Partec GmbH). The control for the tested samples was material collected from a commercial variety sown in pots in a greenhouse. One thousand nuclei were analyzed in each sample. The results of the measurements of the fluorescence emitted by the fluorochrome-stained DNA in cell nuclei were recorded in the form of histograms (Figure 2). To confirm the homozygosity of the obtained plants, 2 isoenzymatic systems were used: GPI and AAT. In order to isolate proteins from plant material, young leaves were collected from adapted plants growing in a greenhouse. Isolation was performed according to the method described by Weeden and Gottlieb (1980). Electrophoresis was carried out on 10% starch gel according to the procedure described by Gottlieb (1973). For electrophoresis, we used the lithium-boron buffer of Selander et al. (1971), stabilizing the environment of electrophoresis and the separation of proteins on the gel. Visualization of the electropherograms was performed according to the procedure described by Weeden and Gottlieb (1980). We performed statistical analysis using a replication of 1 flask (40 anthers). The number of laid anthers is given in Tables 1 and 2. The data were analyzed using
50
100
150
200
125
Figure 2. Histogram of cytometric analysis for plants with DNA amount equal to 2× chromosomes.
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nonparametric analysis such as the Kruskal–Wallis test (with Conover–Inman post hoc tests) using Statistica v. 8.0. for Windows (StatSoft Inc. Tulsa, OK, USA) 3. Results The first embryos were observed 3 weeks after laying out anthers onto the media. They appeared on the outside of the anthers, with no evidence of callus formation. The process of embryo formation lasted for a few weeks. Both polyamines, whether used separately or in combination, had an influence on increasing the number of androgenetic embryos obtained in the Kazan F1variety (Table 1). The greatest influence was achieved on the medium supplemented with putrescine. By contrast, in the Narbonne F1 variety (Table 2), no increase in the efficiency of androgenetic embryogenesis was achieved through the application of polyamines. When regeneration of the embryos obtained on all induction media was performed on B5 regeneration medium, the embryos continued to develop and underwent conversion into complete, properly formed plants. Rosettes, incipient rosettes, and secondary embryos also formed. Also evident was the formation of calli in small amounts and occasionally deformations; e.g., calluslike leaves were formed and a callus heel appeared between the above- and belowground parts. No differences were found between the experimental combinations as regards the course of regeneration and the occurrence of the aforementioned deformations. In the experiment on the influence of putrescine on the regeneration of plants from embryos obtained in anther cultures there was a positive effect of the addition of 160 mg putrescine per 1 L of medium. The largest number of rooted rosettes was obtained on this particular medium. Increasing the concentration of putrescine to 322 mg/L resulted in a reduction in the number of plants obtained. In contrast, in the case of the embryos obtained on the induction medium with putrescine, regeneration was most efficient on the regeneration medium supplemented with 0.5 mg putrescine per 1 L (Table 3). The vast majority of androgenetic plants had a doubled chromosome set. Spontaneous diploidization had taken place. The induction medium was found to have no effect on the ploidy of obtained plants. More than 80% of plants were homozygous for GPI and AAT. At the same time, the distribution of homozygotes and heterozygotes among plants from embryos obtained on different induction media varied widely. 4. Discussion As pointed out in Section 1, the available literature does not contain much information on the influence of polyamines on the efficiency of androgenesis in anther
GÓRECKA et al. / Turk J Biol Table 1. Effect of polyamines on the effectiveness of embryo induction in anther cultures of carrot cultivar Kazan F1. Media
No. of cultured anthers
No. of obtained embryos
No. of embryos per 100 anthers
Control
502
349
69.52 c*
Putrescine
430
1044
242.79 a
Spermidine
317
313
98.73 b
Putrescine + spermidine
471
457
97.02 b
*Combinations within the same homogeneous group (having the same letter) do not differ significantly from each other at a significance level of α = 0.05, based on the Kruskal–Wallis test. Table 2. Effect of polyamines on effectiveness of embryo induction in anther cultures of carrot cultivar Narbonne F1. Media
No. of cultured anthers
No. of embryos per No. of embryos 100 anthers Per 100 anthers
Control
472
5
Putrescine
398
5
1.25 a
1.05 a*
Spermidine
475
9
1.89 a
Putrescine + spermidine
471
5
1.06 a
*Combinations within the same homogeneous group (having the same letter) do not differ significantly from each other at a significance level of α = 0.05, based on the Kruskal–Wallis test.
cultures. However, among the relevant papers found, most of the information concerns the effects of 2 polyamines, spermidine and putrescine, on this process. According to Garrido et al. (1995), the synthesis of polyamines is required for the formation of embryos and not for the induction of embryogenesis in isolated microspore cultures of Nicotiana tabacum (L.). Kumar et al. (2004) used putrescine and spermidine as an addition to the induction medium in experiments with anther cultures of cucumbers. Both these polyamines positively affected the number of obtained embryos, but the most embryos per 60 laid out anthers were obtained with spermidine at a concentration of 200 µM for 2 tested cultivars of cucumber. Dewi et al. (2004) obtained in anther cultures of barley an improvement in all tested parameters: an increase in the number of calli, number of responding calli, number of green and total plants, ratio of green plants to the number of responding calli, and percentage of green plants to the number of anthers inoculated under the influence of all 3 polyamines, putrescine, spermidine, and spermine. However, putrescine was more efficient than spermidine and spermine in increasing callus induction in green plant regeneration in rice anther cultures of Taiper 309. The best
concentration to increase green plant regeneration was 10–3 M. Chiancone et al. (2006), in their studies of androgenesis in clementines using putrescine, spermidine, and a mixture of both, achieved an improvement in the efficiency of androgenesis under the influence of spermidine. By contrast, putrescine did not positively affect the formation of embryos in Citrus clementina. Hema and Murthy (2008) found that the addition of the polyamines putrescine and spermidine to the B5 induction medium with 10 µM 2,4D, 2 µM kinetin, and 0.2 M sucrose enhanced the rate of embryogenesis in anther cultures of niger (Guizotia abbsinica). Dewi and Purwoko (2008) used putrescine as an addition to the induction medium and showed that 10–3 M putrescine had an effect on increasing the efficiency of androgenesis in anther cultures of rice subspecies indica, recognized as a difficult species for androgenesis induction. Some authors have achieved improvement in the efficiency of androgenetic embryogenesis by using various compounds that prevent aging in anthers and ethylene inhibitors, which include polyamines. In a study by Tiainen (1992) on anther cultures of the potato (Solanum tuberosum (L.)), reducing agents, ascorbic acid, and L-cysteine
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GÓRECKA et al. / Turk J Biol Table 3. Effect of polyamines on regeneration from androgenetic carrot embryos in cv. Kazan F1. Kind of media Induction
Regeneration
Rosettes with roots
B5 I 100
B54 reg
55 c*
B5 I 100
B5 + 0.5P
53 c
B5 I 100
B5 + 160P
104 a
B5 I 100
B5 + 322P
51 c
B5 I 100 + P
B5 + 0,5P
85 a
B5 I 100 + P
B5 + 160P
63 b
B5 I 100 + P
B5 + 322P
63 b
Induction media B5 I 100: medium B5 (Gamborg et al., 1968), 100 mg dm–3 L‑serine, 500 mg dm–3 L‑glutamine, NAA, and 2,4-D at 1 mg dm–3 each, and 100 g dm-3 sucrose. B5 I 100+P: medium B5, 100 mg dm–3 L‑serine, 500 mg dm–3 L‑glutamine, NAA, and 2,4-D at 1 mg dm–3 each, and 100 g dm–3 sucrose and 322 mg dm–3 putrescine. Regeneration media B54reg: medium B5 (Gamborg et al., 1968) without hormones and amino acids, with 20 g dm–3 sucrose. B5+0.5P: medium B5 without hormones and amino acids, with 0.5 mg dm–3 putrescine. B5+160P: medium B5 without hormones and amino acids, with 160 mg dm–3 putrescine. B5+322P: medium B5 without hormones and amino acids, with 322 mg dm–3 putrescine. *Combinations within the same homogeneous group (having the same letter) do not differ significantly from each other at a significance level of α = 0.05, based on the Kruskal–Wallis test.
prevented browning of anther cultures and significantly stimulated embryogenesis. Embryogenesis was also promoted by the use of the ethylene inhibitor AgNO3, and by the polyamines spermidine and putrescine. Cho and Kasha (1992), in a study of barley anther cultures, noticed anther browning. They linked this finding to the low efficiency of cultures and decided that the rapid aging of anthers inhibited the formation of embryos from
microspores. In their opinion, this is one of the reasons for the small number of embryos obtained in anther cultures. Polyamines retard aging, and thus may contribute to increasing the efficiency of androgenetic embryogenesis. In the latest publication among those discussed here, Redha and Suleman (2011) obtained an increase in the number of what they called embryo-like structures (ELSs) under the influence of spermidine, putrescine, and spermine, which were used to treat anthers for 1, 3, and 6 h. Although the exogenous application of polyamines to anthers improved the production of ELS and green plants, the effects of putrescine, spermidine, and spermine were dependent on the genotype and the duration of the pretreatment of anthers with polyamines. In our study, the addition of putrescine, spermidine, and a mixture of the 2 to the induction medium had a beneficial effect on the number of embryos obtained in the Kazan F1 variety. This was particularly evident in the case of putrescine. By contrast, polyamines did not have a positive effect on the Narbonne F1 variety. As with most life processes and the effects of external factors on them, a strong influence of genotype has also become apparent here. Purwoko et al. (2001) found a positive effect of polyamines on the regeneration of green plants from embryos obtained in anther cultures of rice subspecies japonica. Dewi et al. (2006) showed that 10–3 M putrescine produced the best effect on the regeneration of androgenetic plants of rice in both the japonica and indica subspecies. Our study also found that the addition of putrescine to the regeneration medium had a beneficial effect on the regeneration process in plants of the Kazan F1 variety, both in the case of embryos obtained on the control induction medium and on the medium with putrescine. However, the concentrations of putrescine in the regeneration media that produced beneficial effects were different for the 2 types of embryos. Acknowledgment This research was funded by the National Centre for Research and Development, Project No. N R12001106.
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