Published online 19 March 2008
Published in Crop Sci 48:651-655 (2008)
© 2008 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
Micropropagation of Field-Grown Perennial Teosinte from Node Culture
J. Zalea,d,*,
L. Ohnoutkováa,c,d,
D. Westa,
C. Samsa,
J. Biggerstaffb and
M. Le Puilb
a Dep. of Plant Sciences, Univ. of Tennessee, Rm. 252 Ellington, 2431 Joe Johnson Dr., Knoxville, TN 37996-4561
b Univ. of Tennessee, Center for Biomarker Analysis, 10515 Research Dr., Suite 300, Knoxville, TN 37932-2575
c present address: Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Sokolovská 6, 77200 Olomouc, Czech Republic
d contributed equally to this work
* Corresponding author (jzale{at}utk.edu).
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ABSTRACT
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Propagation of perennial diploid teosinte, Zea diploperennis Iltis, Doebley and Guzmán (2n = 2x = 20), and tetraploid teosinte, Z. perennis (Hitchc.) Reeves and Magelsdorf (2n = 4x = 40), is limited by seed availability. For plants grown in the field, a photoperiod response delays flowering until fall, and seeds may not reach maturity. The objective of this study was to develop an in vitro micropropagation protocol for field grown teosinte. Shoot proliferation was induced from nodes split longitudinally and plated on Murashige and Skoog's (MS) medium supplemented with 5 µM 6-benzyl amino purine and 3% (w/v) sucrose. Both species possess a single axillary bud that generated multiple plants on division. Rooting of shoots was achieved in half-strength MS medium with and without the addition of 0.4 µM indole-3-butyric acid and 2% (w/v) sucrose. Nodes and younger branches of the tetraploid generated significantly more plants than those of the diploid.
Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid • BAP, 6-benzyl amino purine • IBA, indole-3-butyric acid • MS, Murashige and Skoog • NAA, napthaleneacetic acid • PPM, Plant Preservation Mixture • TIFF, tagged image file format
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INTRODUCTION
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ZEA DIPLOPERENNIS ILTIS, DOEBLEY AND GUZMÁN and Z. perennis (Hitchc.) Reeves and Magelsdorf are warm-season, C4 perennial grasses that are wild relatives of Z. mays (Doebley and Iltis, 1980; Iltis and Doebley, 1980). Both are highly tillered, with elongated axillary shoots tipped by male infloresences arising from nodes and short secondary axillary branches tipped by female infloresences (ears) (Shimizu-Sato and Mori, 2001). These grasses are commonly referred to as teosinte (Iltis and Doebley, 1980). Z. diploperennis is a diploid (2n = 2x = 20) that grows 2- to 2.5-m tall, whereas Z. perennis is a tetraploid (2n = 4x = 40) that grows 1.5- to 2-m tall (Doebley, 2003; Doebley and Iltis, 1980). The teosintes originated in the state of Jalisco, Mexico, at relatively high altitudes (Doebley, 2003).
The diploid, tetraploid, and domestic corn x teosinte hybrids are being evaluated as bioenergy crops at the University of Tennessee. Propagation of these species from seed is limited by seed availability. Furthermore, seed may not mature in the field because plants exhibit a photoperiod response that delays flowering until fall.
Previous studies have examined the production of diploid teosinte via a callus phase. Prioli et al. (1984) excised explants from subapical shoot meristems of Z. diploperennis, plated them on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D) to induce callus formation, and generated plants from calli. Later, this same group reported high frequencies of plant regeneration with this same system (Sondahl et al., 1984). There are no reports of an in vitro micropropagation system for Z. diploperennis or Z. perennis. A micropropagation protocol will permit large numbers of clones, identical to the parent, to be produced and transplanted to the field for biomass evaluation.
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MATERIALS AND METHODS
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Flow Cytometry
The ploidy levels of Z. diploperennis and Z. perennis were verified relative to samples of Z. mays using an LSR II flow cytometer from BD Bioscience (San Diego, CA) at the Center for Wildlife Health at the University of Tennessee. Leaf samples were collected from the field, and 20 mg of newly formed leaves were placed in a Petri dish with 1 mL of OTTO I buffer (Loureiro et al., 2006) and finely chopped with a razor blade. The leaf nuclei were filtered through a 30 µm nylon mesh into 5-mL test tubes to which 2 mL of OTTO II (Loureiro et al., 2006) buffer was added. The nuclei were stained using 1 mL propidium iodide (Fisher Scientific, Hampton, NH; Dolezel and Gohde, 1995). The relative fluorescence intensities were detected by blue argon laser with an excitation at 488 nm (10,000 nuclei per sample). The data were analyzed using DiVa software (BD Bioscience) and represented as pg of DNA for each teosinte species relative to pg of DNA for domestic corn.
Micropropagation
Seeds of Z. diploperennis and Z. perennis were obtained from North Central Regional Plant Introduction Station in Ames, IA, and sown in space-planted plots at the University of Tennessee, East Tennessee Education and Research Center, Knoxville, TN, in 2004. Of the plants that overwintered, five preanthesis axillary branches with 6 to 7 nodes from one plant of Z. diploperennis and one plant of Z. perennis were collected from the field. Younger branches have fewer differentiated nodes. From these axillary branches, five nodal segments, 3-cm long, were dissected. The uppermost nodes from which the male inflorescence would arise were not dissected. The nodes were washed in 70% ethanol for 5 min, 70% bleach for 10 min, 1% mercuric chloride for 15 min, and five consecutive sterile water washes, 5 min per wash, with continuous agitation. Explants, 1.5- to 2.5-cm long, were aseptically dissected from the primary nodes, split longitudinally to form half nodes, and plated flat side down on MS (Murashige and Skoog, 1962; Fisher Scientific, Hampton, NH) medium (pH 5.8) containing 5 µM 6-benzyl amino purine (BAP), 3% (w/v) sucrose, plus 1 mg L–1 thiamine and 500 mg L–1 glutamine, solidified with 7 g L–1 agar (Fisher Biotech Granulated Agar, Fairlawn, NJ); 4 mL L–1 of Plant Preservation Mixture (PPM; Plant Cell Technology, Inc., Washington, DC) was included as an antimicrobial agent. Ten half nodes from each branch were plated in one container. In total, 55 nodes (110 half nodes) of Z. diploperennis and 40 nodes (80 half nodes) of Z. perennis were plated. Cultures were incubated at 26 ± 2°C under 60-W cool white fluorescent bulbs (Philips, Rock Hill, SC) providing light at 60 µmol m–2 s–1 for a 12-h photoperiod. Subculturing was performed every 3 wk by splitting the proliferating shoots and transferring them to fresh MS shoot medium. There were two consecutive subcultures to fresh MS shoot medium from the initial medium, and with each passage, the PPM concentration was halved, from an initial concentration of 4 mL L–1.
Proliferating shoots were transferred to rooting medium consisting of half-strength MS (pH 5.8) containing 2% (w/v) sucrose plus 1 mg L–1 thiamine, 500 mg L–1 glutamine, and 0.4 µM indole-3-butyric acid (IBA; Fisher Scientific, Hampton, NH), solidified with 7 g L–1 agar, for four successive subcultures at two- to three-week intervals. The roots of generated plants were treated in 2% 3-indolebutyric acid (Rhizopon 2, Hazerswoude, the Netherlands) according to the instructions, transplanted to 15.2-cm (6-in) pots containing Sunshine Mix Number One (Sungro, Bellevue, WA), and grown in the greenhouse at 24 ± 6°C, after which time humidity and light intensity were not controlled.
Histology and Widefield Deconvolution Microscopy
To illustrate the axillary buds, diploid and tetraploid teosinte nodes less than 1 cm in diameter were dissected and fixed in Histochoice (Fisher Scientific, Hampton, NH) for 48 h before paraffin embedding (Trigiano et al., 2005). The nodes were dehydrated in a serial series of isopropanol solutions (30, 50, 75, 95, and 100%) at 12 min per solution. The samples were infiltrated by adding paraffin pellets periodically over the next several hours at 60°C, and afterward, the isopropanol was allowed to evaporate. The infiltrated nodes were cast into paraffin blocks and, after cooling, sectioned into 12-µm-thick longitudinal slices using a microtome (Reichart-Jung, Heidelberg, Germany). The sections were immobilized on microscope slides (7.62 by 2.54 cm [3 by 1 in]) (Fisher Scientific, Hampton, NH) by heating to 40°C overnight and stained using fast green and safranin (Sass, 1958). The slides were washed, dried at 40°C, and viewed at 40X total magnification (4X objective). Digital images were taken using a Nikon Eclipse 80i mounted with a Nikon digital imaging head (Nikon, Melville, NY) and saved as Tagged Image File Format (TIFF) files.
Widefield deconvolution microscopy and image analysis were performed at the Center for Biomarker Analysis, University of Tennessee. Teosinte nodes less than 1 cm in diameter were harvested from the field and stored at 4°C until processed. One-millimeter cross-sections of nodes were cut and placed onto a microscope slide (3 x 2.54 cm; Fisher Scientific, Hampton, NH). Node cross-sections were placed in a sterile Petri dish (Fisher Scientific, Hampton, NH), submerged in a drop of Giemsa stain (0.4% in 70% methanol; Sigma-Aldrich, St. Louis, MO) using a sterile transfer pipet, and allowed to incubate at room temperature in the dark for 20 min. The cross-sections were then washed gently with 1X phosphate buffer solution three times to remove excess stain. Node cross-sections were observed at 100X total magnification (10X objective) using an Olympus BX61 automated microscopy platform (Olympus, Center Valley, PA). Stained cross-sections were observed with a FITC filter cube (excitation: 496 nm; emission: 520 nm; Leica, Solms, Germany) for better brightness and contrast. Images were acquired with a CCD camera (VDS Vosskühler, Osnabrück, Germany) and transferred in TIFF format to an image analysis platform for further analysis. AutoDeblur and Image-Pro Plus softwares (Medium Cybernetics, Silver Spring, MD) were used to deconvolve (i.e., remove the blurriness and correct the images for optical aberrations) and enhance acquired images and reveal structures of interest.
Data Analysis
Descriptive statistics and ANOVAs were used to analyze and compare the numbers of plants generated between the two species in micropropagation. For quantitative data analyses, the data for the numbers of plants produced per branch had an exceedingly wide range; therefore, logarithmic transformations were used to normalize the distribution (Steel and Torrie, 1980). A simple, randomized ANOVA for a mixed-effects model with branches being the random effect, nested within species, was used to test for significant differences among the branches (Minitab 14). A simple, randomized ANOVA for a fixed-effects model with unequal cells using the transformed data of the number of plants produced per node was used to test for significant differences between the species (Minitab 14).
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RESULTS AND DISCUSSION
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Seeds of diploid and tetraploid teosinte, originally collected in Mexico and maintained at the North Central Regional Plant Introduction Station in Ames, IA, were planted in Knoxville in 2004. Flow cytometry was used to verify ploidy levels before micropropagation experiments commenced in 2005. Leaf samples of the diploid and tetraploid were tested in flow cytometry using domestic corn as a reference. The DNA content of Zea diploperennis relative to Zea mays (pg DNA Z. diploperennis per pg DNA Z. mays) was 1.0, whereas that of Zea perennis to Z. mays was 1.8, validating that the genome size of Z. perennis was almost twice the size of Z. diploperennis and Z. mays.
Three experiments were conducted during the summer of 2005 to determine optimal concentrations of BAP (0, 3, and 5 µM) for multiplying plants from field-grown teosinte nodal explants. In these experiments, over 500 explants were tested at different concentrations of BAP, and MS medium supplemented with 5 µM BAP was the most effective treatment (data not shown). In banana [Musa X paradisiaca (L.)], MS medium supplemented with 5 µM BAP also produced the greatest number of shoots per explant (Rahman et al., 2002). In this early teosinte work, there were persistent problems with fungal contamination that were not resolved until PPM was incorporated in the medium. Thereafter, PPM was included in all medium.
The objectives of the current experiment were to compare the responses of the two species on MS medium supplemented with 5 µM BAP and to produce plants for field experimentation. Nodes from preanthesis axillary branches with 6 to 7 nodes were used in micropropagation experiments (Fig. 1
). There were significantly more plants generated from the nodes of the tetraploid species than the diploid species, even though more nodes were plated for the latter (Table 1
). In less than 20 weeks, nodes of the tetraploid produced 452 plants, whereas nodes of the diploid produced 60 plants. Multiple shoots were produced from some of the nodes of both species after division. Many of the nodes derived from the older axillary branches did not produce any plants whatsoever. This may have been because the plants were beginning to senesce, given the late fall sampling. In addition, no plants were produced if the explant did not contain an axillary bud. Thirty nodes of the diploid were productive, whereas 15 nodes of the tetraploid were productive. This method of clonal propagation was effective for the diploid but highly efficient for the tetraploid. Axillary bud outgrowth is promoted by cytokinin levels relative to auxin (Shimizu-Sato and Mori, 2001), and the tetraploid responded more vigorously to 5 µM BAP than the diploid.
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Table 1. The number of explants (nodes) plated, productive explants (nodes), the number of plants produced, and the mean number of plants produced per node and per branch for Zea diploperennis and Z. perennis.
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There were significant differences in the number of plants produced among the branches, with younger branches producing the most plants (Table 1). The youngest branch of the tetraploid produced 366 plants, whereas the youngest branch of the diploid generated 18 plants. In general, an equal number of plants were generated from each node on a branch.
Longitudinal sections of the nodes showed a single axillary bud in both species (Fig. 2
). No definitive conclusions about differences in the size of the axillary buds can be made at this time because of inherent variability in sampling. In culture, these axillary buds were divided and multiple shoots developed in response to 5 µM BAP. There was no evidence of accessory buds in either species. Cross-sections of both the diploid and tetraploid nodes viewed with widefield deconvolution microscopy illustrated the one leaf trace per node amidst the vascular bundles (Fig. 3
). Images from one planar cross-section, viewed with widefield deconvolution microscopy, were used to form a composite image showing the scattered vascular bundles of the node (Fig. 4
).
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Figure 2. Longitudinal section of Zea perennis showing one axillary bud per node. MB = main branch; AX = axillary branch. Scale bar 20 µm.
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Figure 3. Cross-section of a Zea diploperennis node showing a single leaf trace (LT). VB = vascular bundles; EP = epidermis. Scale bar 50 µm.
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Figure 4. Composite collection of cross-sections at a node showing scattered vascular bundles of Zea diploperennis using widefield deconvolution microscopy. Scale bar 100 µm.
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The two species differed in growth rates in culture, in growth from seed, and in morphology in the field. The vigorous growth of the tetraploid was not only evident in culture (Fig. 5
) but also in the establishment of young plants from seed in a controlled environment (data not shown). The tetraploid consistently produced more tillers at an earlier stage when grown from seed at 25 ± 2°C, although both species produced the same number of tillers in the field (data not shown). Other morphological differences between the two species were apparent in the field. The mean diameter of the diploid branches was 1.3 cm (SD ± 0.24; n = 24), whereas the mean diameter of the tetraploid was 0.8 cm (SD ± 0.15; n = 24). Similarly, the branches of the diploid remain erect, whereas those of the tetraploid tended to lodge (data not shown).
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Figure 5. The differences in the number and vigor of multiple shoots between (A) Zea diploperennis and (B) Z. perennis grown on Murashige and Skoog's medium with 5 µM 6-benzyl amino purine and 3% w/v sucrose.
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Rooting of generated shoots occurred in half-strength MS with or without IBA at 0.4 µM, but IBA was routinely added to the medium for all subcultures. Rooting success was 85% for both species. Plantlet transfer to potting medium, acclimation, and finally, transfer to the field are the next crucial steps in successful plant propagation (Trigiano and Gray, 2005). A 95% rate in successful greenhouse transplantation and acclimation of plants was achieved. The plants reproduced in this study will be transplanted to the field to monitor growth characteristics.
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NOTES
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All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.
Received for publication February 13, 2007.
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REFERENCES
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ABSTRACT
INTRODUCTION
