Effect of low temperature on in vitro androgenesis of ...

In Vitro Cell.Dev.Biol.—Plant DOI 10.1007/s11627-015-9665-1

BIOTECHNOLOGY

Effect of low temperature on in vitro androgenesis of carrot (Daucus carota L.) Waldemar Kiszczak & Urszula Kowalska & Agata Kapuścińska & Maria Burian & Krystyna Górecka

Received: 26 February 2014 / Accepted: 8 January 2015 / Editor: John Forster # The Society for In Vitro Biology 2015

Abstract Low temperature (4°C) is often applied to donor plants to induce androgenesis in subsequent anther culture. Material for Daucus carota L. (carrot) anther cultures was collected after 9, 12, and 21 d of cool treatment. The most effective treatment proved to be the use of 4°C for 12 d, inducing 24.3 embryos per 100 anthers. Plants were regenerated from embryos, adapted, and then their ploidy and homozygosity were assessed. The analysis of ploidy performed by flow cytometry revealed that all plants obtained through androgenesis contained the amount of DNA corresponding to 2x chromosomes. When assessed for homozygosity, the population was found to consist mainly of homozygotes, 77.8% for glucose-6-phosphate isomerase (PGI, EC 5.3.1.9) and 75.0% aspartate aminotransferase (AAT, EC 2.6.1.1) which indicated the gametic origin of those plants. Distribution of homozygotes and heterozygotes did not depend on the applied thermal shock. Keywords Carrot . Androgenesis . Anther culture . Thermal shock . Ploidy . Homozygosity

Introduction Anther cultures and isolated microspore cultures have been widely used to generate novel genetic variation in plants. This approach is based on the phenomenon of androgenesis, where a sporophyte with the gametic number of chromosomes is a product. As a result of endogenous factors (i.e., spontaneously), or exogenous factors such as colchicine, the chromosome W. Kiszczak : U. Kowalska : A. Kapuścińska : M. Burian : K. Górecka (*) Research Institute of Horticulture, Konstytucji 3 Maja 1/3 str., 96-100 Skierniewice, Poland e-mail: [email protected]

number is doubled (Islam and Tuteja 2012). Doubled haploid lines are widely used both in basic research and breeding programs. The creation of diploid homozygotes and doubled haploid lines can be used in heterotic breeding, a process that requires parental components that will provide a sufficiently high level of heterosis in F1 generation. Androgenesis was induced in more than 250 species of plants in vitro, but development of models of gametic embryogenesis was only successful in four species (Seguí-Simarro and Nuez 2008). Normally, male reproductive cells develop into pollen grains, but as a result of various stimuli, a sporophyte can be induced to switch development pathways resulting in an embryo, and then regenerated into a haploid plant (Bajaj 1990; Touraev et al. 1997; Wang et al. 2000; Shariatpanahi et al. 2006). In plants, the capacity and efficiency for androgenesis is determined by endogenous and exogenous factors. Low or high-temperature stress, osmotic and starvation pressures affect anthers in the early stages of development and are considered to be primary exogenous determinants of androgenesis (Bhojwani and Dantu 2010). Maraschin et al. (2005) suggested that signaling pathways induced by various stresses converge resulting in the same response in plants. Therefore, apart from selection of a genotype with embryogenic capacity and a suitable microspore development stage (Górecka et al. 2005a, b), it is important for most plants to subject anthers, plant parts, or whole plants to the action of a single stress or several kinds of stress to induce embryo formation. The most commonly discussed abiotic stress factors include, among others, exposure to high or low ambient temperature that causes thermal shock (Ślesak and Miszalski 1999). According to Szczuka et al. (2006), exposure of microspores to low temperature after mitosis causes the breakdown of microtubules that determine asymmetric division of nucleus. Touraev et al. (1997) described the formation of microspores where an asymmetric division of the nucleus results in cessation of the growth of the vegetative cell at the beginning

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of phase G1, while the generative cell divides into two sperm cells. Application of low temperature causes disruption to development of the vegetative cell by deregulating the mechanism that blocks the stagnation in phase G1 and the synthesis of specialized regulatory proteins occurs, which are responsible for the transition of cell to S and G2 phase. Thus, stimulated divisions cause induction of androgenesis in anther cultures and isolated plant microspore cultures. In corn, Uváčková et al. (2012) observed the first divisions on the third day of anther cultures. They report that a simultaneous increase in expression of 19 new regulatory proteins under stress conditions. Lulsdorf et al. (2012) emphasized the role of exogenous abscisic acid and other phytohormones in the induction of androgenesis, which decrease under low-temperature stress. They stated that content reduction is a necessary factor affecting androgenesis in recalcitrant species. The pioneers in the use of low temperature, Nitsch and Norreel (1973), were able to increase the efficiency of androgenesis in Datura inoxia flower buds. Thereafter, treating plant material with a cold shock of 4°C has since become a commonly applied for many species, and its effect depends on the genotype (Keller and Armstrong 1977; Keller and Armstrong 1983; Pauk et al. 1991; Krzyżanowska and Górecka 2008). Often temperature is applied in combination with other factors such as high levels of sugar in induction medium (Kasha et al. 1990; Ochatt et al. 2009). In a pioneering study of androgenesis in carrot, Andersen et al. (1990) applied low-temperature shock directly on truncated umbels. They increased the number of responding anthers from 2.4 to 3.7% by placing the umbels for 1 d at low temperature. Embryos and callus appeared on anthers, from which haploid carrot plants were regenerated. Three years later, Hu et al. (1993) regenerated 18 carrot plants using the same method. Matsubara et al. (1995) also studied androgenesis in carrot anthers and isolated microspore cultures, from which embryos were obtained and regenerated. Several hundred androgenetic plants were obtained in anther cultures by Tyukavin et al. (1999). Ferrie (2007) obtained embryos in carrot isolated microspore cultures and regenerated plants but did not detail the methodology. Górecka et al. (2005a, b; 2009a, b) described the impact of various factors on the efficiency of androgenesis in carrot anther cultures but did not conduct research on application of low temperatures for inducing androgenesis in carrot. Our studies were aimed at testing the effect of this factor on androgenesis and determining the optimal duration of low-temperature application (4°C) on donor plants.

Materials and Methods Production of donor plants. Field-grown carrot roots of Narbonne F1 variety were harvested and placed in boxes in

alternate layers of acidic peat (pH 4.0–5.5). For vernalization, they were placed in a cooling chamber at a temperature of 4°C for 3 mo after which roots were planted, two at a time, into plastic containers with a substrate consisting of 1 part peat and 2 parts sand. To ensure the proper pH, chalk was added to the substrate at 8 kg m−3 of substrate, and Azofoska (Inco Veritas, Group Inco S.A. Warsaw, Poland) fertilizer was applied as a source of macro- and micronutrients, at 1.2 kg m−3 of used peat, containing 13.6% N, 1.9% P, 16.0% K, and trace elements: Cu, Zn, Mn, B, Mo. Planted roots were placed in a growth chamber under controlled growth conditions at a temperature of 20/16°C day/night, with 16 h illumination with a light intensity of 30 μmol m−2 s−1. After 1 mo growth, plants were watered at 14-d intervals with 0.3% solution of Hydrovit 300 (Hydrokomplet S.C, Nowa Wieś k/Częstochowy, Poland) containing 2.20% N-NO3, 0.45% P, 2.26% K, 1.32% Ca, 0.49% Mg, and microelements such as Fe, Mn, Cu, Zn, B, and Mo. Low-temperature shock treatment. Six heterozygous plants with undeveloped inflorescences were placed in a cold store at 4°C. Then, after 9, 12, and 21 d from the time of placing, flower buds were collected from 12 developed umbels containing anthers at the optimum stage of microspore development. Anthers were also collected from plants placed in a growth chamber. These plants were constituted as control. Next, buds were sterilized for 2 min in 70% ethanol and rinsed twice with sterile distilled water; then, anthers were isolated and placed in 100 mL Erlenmeyer flasks containing about 30 mL of a medium for the induction of androgenesis. The used medium was B5 (Gamborg et al. 1968), modified by Keller and Armstrong (1977) and applied by Andersen et al. (1990) for carrot anther cultures, containing 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthaleneacetic acid (NAA) each at 0.1 mg L−1, Lglutamine at 500 mg L−1, L-serine at 100 mg L−1, and sucrose at 100 g L-1. Flasks with anthers were placed in the dark at 27°C. When embryos had appeared, flasks were exposed to continuous light with an intensity of 30 μmol m−2 s−1 while maintaining the same temperature (Fig. 1A). Plant regeneration and adaptation. Green embryos (Fig. 1B) were described, counted, and transferred onto B5 regeneration medium without hormones with 20 g L-1 sucrose. The phytotron temperature was 20°C, and lighting was provided for 16 h at 30 μmol m−2 s−1 (Fig. 1C). Plants that developed roots in regeneration process were transplanted into multipots filled with a substrate of sand and peat (3:1; v/v) with the addition of the multicomponent complex fertilizer ‘Azofoska’(Inco Veritas, Group, Inco S.A. Warsaw, Poland) 13.6% N, 1.9% P, 16.0% K, and microelements such as Mg, Cu, Zn, Mn, B, and Mo—at 1.25 kg m−3 of peat, and a dose of chalk at 8.0 kg m−3. Adaptation was carried out under the conditions of high humidity in a small plastic tunnel placed in a growth chamber at a temperature of 20°C during the day and 18°C at night, with

EFFECT OF LOW TEMPERATURE ON IN VITRO ANDROGENESIS OF CARROT Figure 1. A and B Androgenetic embryos in a flask. C Carrot plants during regeneration. D Adapted androgenetic carrot plants in multipots.

16 h of light at an intensity of 30 μmol m−2 s−1 (Fig. 1D). Adapted androgenetic carrot plants were subsequently assessed in terms of ploidy and homozygosity. Assessment of ploidy. Ploidy was determined indirectly by means of a flow cytometer. Galbraith (1984) procedure was used, with modifications. Because of the high content of phenols in plant material, which exhibit inhibitory activity against DNA staining by fluorochrome, it was impossible to use the standard buffer and thus 0.1% (w/v) polyvinylpyrrolidone (PVP-40) was used. Approximately 20 mg of young leaves were crumbled with a razor blade in a petri dish containing 2 mL of CyStain® DNA (Partec GmbH) cell lysis buffer, and polyvinylpyrrolidone (PVP-40). After adding fluorochrome 4′, 6-diamidino-2-phenylindole (DAPI) at 0.1 mg mL−1, samples were filtered through a 50-μm nylon filter (Partec ‘Cell Trics’, Partec GmbH, Münster, Germany). Thus, prepared samples were analyzed by means of a Ca II flow cytometer (Partec GmbH, Münster, Germany). The control for tested samples was material collected from a commercial variety sown into pots in a greenhouse. In each sample, 1000 nuclei were analyzed. Results of the measurements of the fluorescence emitted by fluorochrome-stained DNA in cell nuclei were recorded in the form of histograms. Assessment of homozygosity with glucose-6-phosphate isomerase (PGI, EC 5.3.1.9) and aspartate aminotransferase (AAT, EC 2.6.1.1). Donor plants and androgenetic plants with a double set of chromosomes were tested to confirm homoand heterozygosity. Two isoenzymatic systems were analyzed: PGI and AAT. Only plants which were heterozygous in respect of both isoenzymatic systems were used as donors for anther cultures.

Samples for the tests consisted of young leaves of adapted plants growing in a greenhouse. Leaves were crushed in the tris-maleate extraction buffer (pH 8.0) containing 10% glycerol (w/v), 10% polyvinylpyrrolidone (PVP-40) (w/v), 0.5% Triton X-100 (w/v), and 14 mM β-mercaptoethanol. Electrophoresis was performed on a 10% (w/v) starch gel according to Gottlieb (1973), which contained 15 mL of a lithium-boron buffer (1.20 g L−1 lithium hydroxide, 11.89 g L−1 boric acid, 125 mL Tris pH 8.4, and 16.2 g of potato starch. Separation of enzymes was performed according to the method by Selander et al. (1971). For this purpose, the lithium-boron buffer provided optimum conditions for separation and stabilization of proteins, at 300 V and 50 mA. For visualization of PGI isoenzyme polymorphism, the method by Weeden and Gottlieb (1980) was used, so the gels were dipped in staining solution and placed in the dark at room temperature. Visualization for AAT polymorphism was performed by dipping gel in staining solution and placing in incubator at 37°C. Statistical analyses. Obtained data were analyzed using multivariate statistic ANOVA/MANOVA non-parametric analyses such as the Kruskal and Wallis (1952), at an adopted level of significance of α=0.05. Statistical analyses were performed using Statistica 8.0 software package for Windows (StatSoft Inc. Tulsa, USA).

Results Microspores from anthers cultured in a flask underwent divisions and produced embryos, which became visible on the outside of anthers. Time of embryo emergence depended on

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period of low temperature applied to the donor plants, either 0, 9, or 21 d. Embryos appeared in control combination (0 d) after 87 d. Anthers from plants treated with low temperature for 9 d produced embryos between 91–106 d, low temperature for 12–98 d whereas anthers treated for 21 d generated embryos between 72 and 97 d after culture establishment. The applied low-temperature shock had an effect on embryo formation in anther cultures (Table 1). The most effective stimulant was the use of 4°C treatment for 12 d. From 100 anthers, collected from buds of donor plants treated with that temperature, 24.3 embryos were obtained, whereas only 2.98 embryos were obtained per 100 anthers after 9 d lowtemperature treatment. From umbels of donor plants treated with a low-temperature shock for 21 d, we obtained 15.29 embryos per 100 cultured anthers, whereas by establishing anther cultures without the shock treatment we obtained 3.16 embryos per 100 anthers. Following statistical analyses, significant differences between tested factors were found. From the obtained embryos, 46 plants with typical carrot plant morphology were regenerated with sturdy roots. Also, rosettes without roots and a small number of callus clumps grew in culture. After converting the number of received androgenetic plants with proper structure into 1 laid out embryo 100% such plants regenerated from embryos that appeared on anther, which were collected from donor plants stored in low temperature for 12 and 21 d. When donor plants were kept in 4°C for 9 d, 72% of rooted plants were obtained from one embryo while in control combination, they constituted 92% of regenerated multiplication. Cytometric analysis revealed that all plants obtained through androgenesis contained the DNA corresponding to 2x chromosomes. During conducting isoenzymatic analysis, in case of PGI, two zones of PGI-1 activity of monomorphic character were detected and also PGI-2 of polymorphic character. In this locus, the presence of a single band pattern characteristic for homozygote and a third band pattern characteristic for heterozygote was found (Fig. 2A). In case of AAT, three activity zones were noted on zymogram: AAT-1, AAT-2, and AAT-3. The AAT-1 locus showed Table 1. Effect of the duration of cooling donor plants at +4°C on the efficiency of the induction of androgenesis in anther cultures Duration of cooling (days)

Number of anthers laid out

Number of embryos obtained

Number of embryos per 100 anthers

Number of days until embryo production

Control 9 12 21

348 402 358 353

11 12 87 54

3.16bc 2.98c 24.3a 15.29ab

87 91–106 98 72–97

Combinations within the same homogenous group (having the same letter) do not differ significantly from each other at a significance level of α=0.05. Kruskal-Wallis test

no polymorphism, which characterized the other zones. In the event of this isoenzyme with dimeric structure, AAT-2 and AAT-3 zones should be considered together; therefore, homozygote will be characterized by one band while heterozygote will have 2 or 3 bands (Fig. 2B). We tested 44 plants and found 77.8% PGI homozygotes and 75.0% AAT homozygotes in the population. The rest of population constituted of heterozygotes with respect to both of these isoenzymes. The arrangement of homo- and heterozygotes in experimental combinations was similar.

Discussion In many plant species, such as tobacco (Sunderland and Roberts 1979), pepper (Özkum and Tipirdamaz 2002; Irikova et al. 2011), wheat (Zheng 2003), barley (Lazaridou et al. 2005), rice (Khatun et al. 2012), rye (Mikołajczyk et al. 2012), and other major crop species (Forster et al. 2007; Islam and Tuteja 2012), induction of androgenesis was found to be effective if a low-temperature thermal shock was applied directly on truncated inflorescences, flower buds, donor plants, and anthers. In three species of the family Fabaceae, Ochatt et al. (2009) placed flower buds at 4°C and observed suppression of microspore divisions and a fall in the efficiency of androgenesis by 35% in sweet pea (Lathyrus L.), 30% in alfalfa (Medicago L.), and 25% in pea (Pisum L.) after 1 d of treatment. Increasing the efficiency of androgenesis was gained only by keeping buds for 2 or more days at that temperature. Krzyżanowska and Górecka (2008) applied a low-temperature shock on flower buds of Brussels sprouts for 24 h and did not achieve much improvement in the efficiency of androgenesis. In both the weakly embryogenic variety Maximus F1 and embryogenic Philemon F1, only 0.2% of anthers in anther cultures responded to a 24-h low-temperature application. Andersen et al. (1990) placed truncated umbels of carrot at 5–7°C. They observed an increased efficiency of androgenesis when the umbels remained at 7°C for 1–2 d. Studies on androgenesis in carrot were also conducted by Górecka et al. (2005a, b; 2009a, b). However, during induction of embryos in carrot anther cultures, thermal stresses were not applied. Cultures were conducted at a constant temperature of 27°C, and the rate embryos were induced depended mostly on the genotype. In addition to subjecting various parts of plants to a lowtemperature stress, the effect of the duration of thermal shock application was also studied. For example, triticale was treated at a temperature of 4°C for 2 wk (Pauk et al. 2000), wheat for 7 d (Jauhar 2003), and potatoes for 72 h (Tai and Xiong 2003). In these studies, the authors placed truncated inflorescences or flower buds at a low temperature, which introduced an additional factor to the experimental system associated with the likelihood of oxidative stress caused by truncating a fragment

EFFECT OF LOW TEMPERATURE ON IN VITRO ANDROGENESIS OF CARROT Figure 2. A Example of PGI isoenzyme zymogram with graphic reflection of bands. B Example of AAT isoenzyme zymogram with graphic reflection of bands.

of certain parts of plant, and that is why some authors place entire donor plants at a low temperature. Smýkalová et al. (2009), while conducting research on androgenesis in a relative of carrot, caraway (Carum carvi L.), achieved improvement in the efficiency of androgenesis by placing donor plants at the three-to-four leaf stage at 6°C. Similarly, in our research, positive results were obtained by subjecting whole donor plants to low temperatures, the efficiency of anther cultures dependent on the time of keeping plants at 4°C. Smýkalová et al. (2009) used two different temperatures affecting for different lengths of time on caraway plants developing inflorescences. Temperature of 22°C for 9 d and then 6°C for 19 d were used, which gave an anther response rate of 19%, but the reversal of this temperature and time sequence caused a

decrease in the efficiency of androgenesis and anther response of only 0.17–0.45%. In presented experiment, prolonging the duration of cooling donor plants at 4°C to 21 d allowed to obtain 15.29 embryos per 100 laid out anthers, which is 5 times higher than the control, but it is not the highest value. The efficiency of androgenesis was the highest after 12 d; almost 8 times more embryos were obtained in relation to the control. After 9 days no improvement in the efficiency of androgenesis was observed in comparison with the control. Plants were regenerated from the embryos obtained in our research. Regeneration progressed through direct conversion of embryos into complete rooted plants; also, rosettes without roots were obtained, as well as a small number of callus lumps. Möllers and Iqbal (2009) reported that only very few

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androgenetic embryos of rape regenerated directly into plants, with the majority forming secondary embryos, on cotyledons and hypocotyl, from which shoots were regenerated. In caraway, Smýkalová et al. (2009), after obtaining embryogenic callus in anther cultures, first obtained embryos and then regenerated plants. In the same year, Górecka et al. (2009a), in the process of regenerating androgenetic embryos of carrot, obtained, in addition to complete plants, different multiplication variants: rosettes, secondary embryos, and small amounts of callus. On the other hand, Ferrie et al. (2011) reported that in fennel and caraway, belonging to the same family as the carrot, regeneration proceeded along a direct route. In our research, no difference was observed in the course of plant regeneration from embryos obtained in different experimental combinations. Ploidy analysis showed that all adapted plants contained the quantity of DNA corresponding to the diploid number of chromosomes. This indicates that the use of low-temperature shock had no effect on ploidy of plants obtained through androgenesis. Kiełkowska and Adamus (2010), in their experiments with unfertilized ovules of carrot, obtained 97.7% diploids. Similarly, in our previous studies (Kiszczak et al. 2011), we found that among carrot plants obtained through androgenesis, 90% were diploids, and the rest were haploids and tetraploids. Smýkalová et al. (2009), after obtaining 35% caraway haploids in anther cultures, reported that two thirds of the population were diploids and tetraploids. The appearance of diploids in obtained population was explained by the occurrence of spontaneous diploidization. Tyukavin et al. (1999) observed cytological instability of androgenetic embryos and carrot plants during the regeneration process in in vitro cultures that caused changes in the level of ploidy. In analysis of ploidy, Smýkalová et al. (2012) applied the esterase (EST, E.C.3.1.1.1) isoenzymatic system, where, based on a combined analysis of the polymorphic locus Est1 and monomorphic locus Est-2, the distribution in the population of homozygotes were determined, and on that basis, the ploidy level of androgenetic caraway plants were defined. In our studies, isoenzymatic analysis was used to determine homozygosity and confirm the gametic origin of plants derived through androgenesis in anther cultures. This characteristic was assessed on the basis of polymorphic loci Pgi-2 and Aat-2 together with Aat-3. In 1989, Westphal and Wricke used PGI enzyme system to determine homozygotes in carrot. Analysis revealed activity of two one-band allelic variants of Pgi-2 locus with a high and low migration rate indicating the homozygous nature of plant. Allelic interaction of these two subunits results in the appearance of a third band with an intermediate rate of migration. That arrangement is characteristic for heterozygotes. In the present study, a similar image of the isoforms of PGI enzyme was obtained, which allowed to make an error-free distinction between homo- and heterozygotes. Bartošová et al. (2005) analyzed two isoenzymes: acid

phosphatase (ACP, EC 3.1.3.2) and peroxidase (PRX, EC 1.11.1.7), in order to confirm gametic origin and determine homozygosity of flax plant (Linum usitatissimum L.) obtained in anther cultures and through gynogenesis. Authors confirmed the usefulness of isoenzymatic analysis in determining homozygosity and gametic origin of plants by using two systems. In 2010, Kiełkowska and Adamus used PGI isoenzymatic system to analyze homozygosity of diploid regenerates of carrot obtained from unfertilized ovary cultures, receiving 45.9% homozygotes. In our experiment, we obtained 77.8% homozygotes characterized by one-band variant for Pgi-2 and 75.0% for AAT, where the presence of a single band was detected in each of the Aat-2 and Aat-3 loci. Presented variants for two isoenzymes are characteristic for homozygotes and confirm gametic origin of plants obtained through androgenesis. The rest of plants in studied populations were heterozygotes. Distribution of homo- and heterozygotes in experimental combinations was similar. In our previous studies (Kiszczak et al. 2011), using the same isoenzymes to determine homozygosity of carrot plants derived from embryos from anther cultures, similar results were obtained. Ferrie and Möllers (2011), Adamski et al. (2014) reported that already in various experiments on the use of isoenzymes and molecular markers a distortion in segregation of markers in DH population was demonstrated, and this explains the appearance of variants characteristic of heterozygotes at loci Pgi-2. This state can be caused by many factors. In presented studies, in order to thermal shock application, donor plants were submitted to the action of low temperature for 9, 12, 21 d. Low-temperature shock often results in disruption in the course of nucleus divisions during meiosis occurred in pollen mother cells. At that time, in telophase chromosome bridges are formed between telophase and post-telophase nuclei (Szczuka et al. 2006). According to Stimpson et al. (2012), such anomalies can lead to chromosome breaking; after their replication and no separation of sister chromatids, dicentric chromosomes can be created. The different observed disturbances are late chromosomes during second meiotic division. Duplication can appear—doubling of chromosome fragment as a result of asymmetric chromatid sections exchange and sometimes incorrect crossing-over. Kasha et al. (2001); Shim et al. (2006) proved that in microspores reprogrammed on sporophyte development path, there is a fusion of sister nuclei causing spontaneous doubling of chromosomes, and in extreme cases, chromosome reorganization can occur. Oleszczuk et al. (2011) discovered that fusion of non-sister nuclei is possible in meiocyte, where cell wall is not a barrier. At that time, this nearly always will result in generating heterozygotic plant from such microspore. Acknowledgments This research was financed by the National Centre for Research and Development, Poland project no. NR 12001106.

EFFECT OF LOW TEMPERATURE ON IN VITRO ANDROGENESIS OF CARROT

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