Chapter 20

Chapter 20 Marigold Regeneration and Molecular Analysis of Carotenogenic Genes Alma Angélica Del Villar-Martínez, Pablo Emilio Vanegas-Espinoza, and Octavio Paredes-López Abstract Marigold (Tagetes erecta) is an Asteraceous plant of industrial, ornamental and medicinal importance. Tagetes erecta species, popularly known as marigold, is grown as ornamental plant and is adapted to several agro climates. Inflorescences have been utilized as pigment source for food coloring, mainly of poultry skin and eggs. Lutein is the main pigment in marigold flowers. Some carotenoids are well known as provitamin A compounds. There are many reports on carotenoids and their effect on the prevention of certain ocular diseases, ischemic heart disease, strokes, photoprotection, immune response, aging and cancer. Marigold flowers are certainly a good source of carotenoids; they show very different pigmentation levels. This chapter describes the establishment of techniques for plant regeneration, gene expression, pigment extraction and pigment deposition in specific structures of marigold. Key words: Tagetes erecta, Organogenesis, Asteraceae, Gene expression, Flower development, Plastids

1. Introduction Carotenoids comprise a large group of secondary metabolites that are natural pigments present in most of higher plants. They are widely distributed in nature and participate in an important set of reactions in plants; moreover, some of them are the main dietary precursors of vitamin A (1). Genetic engineering provides good possibilities to obtain modified plants to improve pigment accumulation in high producing crops (2). Marigold is an Asteraceous plant, and is being used in traditional Mexican medicine as antiparasitic, antispasmodic and against stomach and liver diseases (3) as antimutagenic and antioxidant (4).

S.M. Jain and S.J. Ochatt (eds.), Protocols for In Vitro Propagation of Ornamental Plants, Methods in Molecular Biology, vol. 589 DOI 10.1007/978-1-60327-114-1_20, © Humana Press, a part of Springer Science + Business Media, LLC 2010

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Flowers are good source of carotenoids for food coloring, mainly of poultry skin and eggs; oleoresin is obtained from chopped flowers at processing industrial plants to be used for commercial purposes (5, 6). The uses of this plant underline the importance of establishing reliable plant regeneration, gene expression analysis and genetic manipulation systems aimed at increasing its carotenoids levels (7–9), by enhancing their concentration in the flowers and, at the same time, generating new cultivars with higher number of flowers per plant. Marigold is also a good model to study carotenogenic process (6). Tagetes erecta flowers accumulate high levels of carotenoids, especially lutein being the main pigment in marigold flowers. Lutein isomer peaks have been identified analysing their retention time and absorbance maxima from spectra (10, 11). There are a few reports on marigold in relation to plant tissue culture; the available studies use a wide range of explant sources, and different kind and combinations of plant growth regulators. However, there are no reports about the establishment of a marigold regeneration system or a cell culture for in vitro pigment production. The whole plant regeneration methodology has been carried out at our laboratory from marigold leaf explants using Murashige and Skoog (MS) medium (7, 12). In higher plants, carotenoids are localized in plastids that, during flower development, are transformed from proplastids into chromoplasts (13). Ultrastructure of plastids from marigold flowers undergoes remarkable changes, including internal organization destruction throughout flower development (14). Subcellular studies on Tagetes erecta callus cells have been done in our laboratory, whereby structures such as cell wall, vacuole, nucleus, nuclear membrane, intercellular spaces, proplastids, chloroplasts and chromoplasts were clearly identified. Organelle organization differences between cell types were evident in quantity and plastid appearance (unpublished). Lutein is synthesized in plastids through non-mevalonate pathway (15, 16). Studies on carotenoid biosynthesis and regulation in different organs are necessary for further genetic modification (17). Participating genes in carotenoid biosynthetic pathway have been cloned from T. erecta; northern blot analysis shows differential expression of analysed genes, and lcy-b gene appears to be highly related to flower development and pigment accumulation (14, 18).

2. Materials 2.1. Plant Material

Seeds of a commercial line of marigold are used. In vitro germinated seeds are utilized as explant source for plant regeneration and greenhouse acclimation. Greenhouse-grown marigold

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plants are sampled during flower development at four stages. The plant tissues are harvested, frozen in liquid nitrogen and stored at −70°C until processed for RNA isolation and pigment analysis. 2.2. In vitro Plant Culture

Murashige and Skoog-prepared salt medium (Sigma-Aldrich) is used with or without added plant growth regulators. Medium is solidified adding Phytagel (Sigma-Aldrich) as gelling agent.

2.3. Blotting Material

Isolated and purified RNA’s are transferred onto nylon Hybond+ membranes (Amersham Biosciences, Brazil); prehybridation and hybridation are carried out with HybrisolR (Oncor, USA) (50% formamide; 10% dextran sulfate; 1% SDS; and 100 mg/ml denatured salmon sperm DNA fragments). Films with radioactive signals are analysed using the Gel DOC 2000 and Quantity One Software V.4 (BIO-RAD, Italy).

2.4. Chromatographic Supplies

A YMC PACK C30 (YMC Inc., USA) reversed-phase column (5 mm; 25 cm × 4.6 mm i.d.) and lutein standard (Sigma-Aldrich, USA) are used for chromatographic analysis; using a HPLC 1,050 (Hewlett-Packard, USA).

2.5. Microscopy Requirements

Osmium tetroxide (OsO4) and propylene oxide (C3H6O) are purchased from Sigma-Aldrich, USA. A MTX ultramicrotome (RMC, USA) is used in order to obtain thin sections, and observations are performed with a JEOL 1010 transmission electron microscope (JEOL Ltd., Japan).

3. Methods 3.1. Bud Induction from Leaf Explants, Plantlet Generation and Plant Adaptation to Greenhouse

1. Seeds are surface sterilized with absolute ethanol for 1 min, 70% ethanol for 5 min, 2% sodium hypochlorite for 15 min and 1% sodium hypochlorite for 15 min. As a surfactant, 0.1% Tween-20 is added to sodium hypochlorite solution. 2. A rinse with sterile deionized water is performed after each treatment. Finally, at least three rinses with sterile deionized water are carried out. 3. Surface sterilized seeds are germinated on hormone-free MS (12) medium containing 30 g/l sucrose and solidified with 3 g/l Phytagel. 4. All cultures are maintained at 25 ± 2°C under fluorescent light (50 mmol/m2s) and 16 h light/8 h dark cycle. 5. Three-week-old plantlets are the explant source. Leaf portions about 0.25 cm2 are placed on MS media containing 17.1 mM IAA and 13.3 mM BA.

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6. After thirteen days, shoots are transferred to hormone-free MS medium for plant elongation and rooting. (See Note 1). 7. The well-developed plantlets are then transferred to pots containing sterile soil and protected with plastic covers, modified on the second and fifth day to decrease the internal humidity. 8. The pots are maintained in a growth chamber for 1 week. A solution of half-strength MS salts and sucrose is added on the second and fifth days. After the eighth day, the plants are moved to a greenhouse to reach their complete development. An example is shown in Fig. 20.1. 3.2. Northern Blot Analysis

1. Total RNA (20 mg) samples are loaded under denaturing conditions on a 1.2%, w/v agarose gel containing formaldehyde. 2. The RNA’s are transferred onto Hybond+ membranes, with hybridizations performed under high stringency conditions. 3. The membranes are incubated for 2 h at 42°C in 20 ml prehybridization solution HybrisolR. 4. RNA on membranes is probed with the full-length cDNA for each carotenogenic gene. 5. Denatured and solution.

32

P-labeled probe is added to hybridization

6. Each hybridization is carried out in HybrisolR at 42°C for 16–18 h using 1–2 ng/ml radiolabeled probe. 7. After hybridization, membranes are transferred as quickly as possible to 50–100 ml washing solution for three washing periods decreasing successively the SSPE concentration: SSPE 4×/SDS 0.1%, SSPE 2×/SDS 0.1%, SSPE 1×/SDS 0.1% (all solutions are in w/v relationship), at room temperature during 20 min each one.

Fig. 20.1. Regeneration process from leaf explant to greenhouse-adapted plants. (a) leaf explants in regeneration medium; (b) emerging bud; (c) plantlet development; and (d) greenhouse-adapted plants.


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8. The membranes are removed from washing solution and then placed into exposure cassette with an X-ray film for a suitable time, typically 5 h. 9. Films with signals are scanned using the Gel DOC 2000 and Quantity One Software V.4. The transcript levels are determined. An example of the results obtained is shown in Fig. 20.2. (See Note 2). 3.3. Analysis of Carotenoid Pigment Content in Marigold Flowers by High Performance Liquid Chromatography

1. Carotenoids are extracted from flowers at four developmental stages. 2. Fresh tissue samples are extracted with HEAT (hexane:absolute ethanol:acetone:toluene, 10:6:7:7 v/v/v/v) and 40% methanolic KOH solution. 3. Separations are carried out with an YMC PACK C30 reversedphase column (5 mm; 25 cm × 4.6 mm i.d.). 4. A solvent gradient is programmed into an HPLC analyser model 1050 as follows: solvent A (methanol) is delivered isocratically from 0 to 6 min; then, a 4 min linear gradient to 5% solvent B (isopropyl alcohol) is applied, followed by 5 min isocratically with 5% solvent B; then, a 10 min linear gradient to 25% solvent B is used followed by a 5 min linear gradient to 50% solvent B, a 10 min linear gradient to 75% solvent B, 25% solvent C (hexane), and finishing with 10 min isocratically with the final mixture. Flower developmental stages 1

2

3

4

Lcy-b

Lcy-e

rRNA Fig. 20.2. mRNA analysis in developing flowers of marigold. Differential expression is observed at different flower developing stages. (1) closed bud; (2) semi-open bud; (3) open flower; and (4) fully open flower.

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5. The column is re-equilibrated between samples for 15 min with solvent A. 6. Pigments are monitored at 450 nm, and their spectra are analysed using a diode array detector (10). 7. Lutein isomer peaks are identified by analysing their retention time and absorbance maxima from spectra as described (10) using a lutein standard. Figure 20.3 shows lutein content from different flowering stages extractions. 1. Small pieces of marigold petals are cut and fixed for 12 h in 4% formaldehyde (from aqueous alkaline hydrolysis at 60°C of paraformaldehyde) and 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 at 4°C. 2. After fixation, samples are washed overnight in 0.25 M sucrose in the same buffer. Post-fixation is carried out in 1% OsO4 in cacodylate buffer for 12 h at 4°C. (See Note 3). 3. Tissue samples are dehydrated through graded (10% steps) ethanol series from 10% to absolute (over OsO4); propylene oxide is the intermediate solvent for infiltration. 4. The first infiltration step is carried out with complete resin diluted with propylene oxide (1:1, w/v) for 24 h in a closed jar with silica gel with moisture indicator. 5. A second infiltration step is carried out with complete undiluted resin in a rotatory shaker for 2–4 h at room temperature.

1000

Lutein mg/g fresh weight

3.4. Transmission Electron Microscopy

800 600 400 200 0 1

2

3

4

Flowering stages Fig. 20.3. Lutein content in different flowering stages of marigold. (1) closed bud; (2) semi-open bud; (3) open flower; and (4) fully open flower.


1

2

3 V

li

500nm

500nm

200nm

g

im

s

r

500nm

li

t

op

or

l ch

chromoplast

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las op lor ch

li t las

r

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om

chromoplast

Fig. 20.4. Transmission electron micrographs of callus and marigold flowers. (1) green callus; (2) yellow callus; (3) closed bud; and (4) fully opened flower. Key: cw cell wall; g grana; im inner membrane; li lipid vesicles; om outer membrane; r ribosomes; v vacuole.

6. After the second infiltration step, embedded samples are transferred to flat embedding molds filled with fresh undiluted resin and are polymerised in a convection oven at 60°C for 36 h. 7. To select samples for TEM observation, 1 mm thick sections are obtained with a glass knife and stained with toluidine blue. From the selected areas, thin sections (70–90 nm) are cut with a diamond knife in an MTX ultramicrotome. (See Note 4). 8. Thin sections are attached to a 300-mesh copper grid, contrasted with lead citrate and uranyl acetate, and observed at 80 kV in a JEOL 1010 transmission electron microscope. An example of results from flower and callus samples is shown in Fig. 20.4. (See Note 5).

4. Notes 1. Plant regeneration efficiency may differ depending on the genotype. 2. There are some considerations about gene expression method – gene architecture, specificity, sensitivity, sample requirements, and costs, among others – because of the technique complexity.

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3. Sample preparation for transmission electron microscopy and probe labeling use dangerous substances as radioactive elements (32P) and poisonous reagents (OsO4); they must be used under restricted security conditions. 4. A method of plant regeneration, a system of cell culture from marigold leaf, the gene expression in marigold of carotenoidbiosynthesis related genes, an ultrastructural method for analysis of structures related to sites of pigment accumulation, and a method for genetic transformation by particle bombardment (8) are now available. 5. Hence, with these basic techniques, marigold may be manipulated by genetic engineering in order to produce new kind of carotenoids or any other specific metabolite. Currently, tools exist to improve the potential traits of marigold in order to obtain flowers with modifications in level and type of containing pigment, thus enhancing its commercial potentiality as an ornamental plant.

Acknowledgments We are grateful to Consejo Nacional de Ciencia y Tecnología (CONACYT), Instituto Polittgécnico Nacional (IPN), and to CONCYTEG-Gto., México for financial support.

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