In Vitro Cell Dev. Biol. 31:65-71, April 1995 © 1995 Society tbr In Vitro Biology 1071-2690/95 $05.00 + 0.00

THIDIAZURON-INDUCED ADVENTITIOUS SHOOT REGENERATION OF SWEETPOTATO (IPOMOEA BATATAS)

RAMANA M. GOSUKONDA, ANANTA POROBODESSAI, ESSIE BLAY, CHANNAPATNA S. PRAKASH, 1 and CURT M. PETERSON

Plant Molecular and Cellular Genetics Lab, School of Agriculture and Home Economics, Tuskegee University, Tuskegee, Alabama 36088-1641 (R. M. G., A. P., E. B., C. S. P.); Department of Botany and Microbiology,

Auburn University, Alabama 36849 (C M. P.)

(Received 25 February 1994; accepted 22 December 1994; editor D. W. Altman)

Adventitious shoots of sweetpotato (Ipomoea batatas L. Lain.) were produced in vitro using a two-stage culture method. Petiole explants were incubated on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (0.2 mg'liter -1) for 3 d, and transferred to MS medium with thidiazuron (0 to 0.4 rag-liter ~). Shoot regeneration was observed in most explants (78.2%) of genotype PI 318846-3 within 28 days when cultured on thidiazuron at 0.2 mg-liter-~. Histological studies of cultured petiole explants showed meristematic activity within cells of vascular bundles and through- out the ground tissue. Explants isolated from apical leaves exhibited higher shoot regeneration frequency than those isolated from the basal portion of the shoot. Leaf lamina explants exhibited lower frequency of regeneration than petiole explants. In contrast to thidiazuron, the use of zeatin riboside, and kinetin resulted in a lower frequency of shoot regeneration although more sweetpotato genotypes could be regenerated using either of these two cytokinins. The sweetpotato plants regenerated using thidiazuron grew vigorously and rooted easily when transferred to the greenhouse.

Key words: biotechnology; organogenesis; plant regeneration; thidiazuron; tissue cuhure.

INTRODUCTION

Sweetpotato (Ipomoca batatas L. Lam.), an important food crop in developing countries, has high yield potential, high nutritive value, and tolerance to a range of agroecological conditions (Woolfe, 1992). There are several reports of in vitro organogenesis in sweetpotato using explants derived from various organs and tissues [e.g., coty- ledons (Xue, 1987), leaf pieces (Carswell and Locy, 1984; Temple- ton-Somers and Collins, 1986), petioles (Hwang et al., 1983; Xin and Zhang, 1987); and roots (Gunckel et al., 1972)]. Apical buds, lateral buds, stem, anther, and storage roots have also been used to regen- erate plantlets [reviewed in Henderson et al. (1983); Kuo (1991)]. Organogenesis has also been induced in swcetpotato through somatic embryogenesis using shoot tips and other explants (Ja~Tet et aI., 1984; Lin and Cantliffe, 1984). Plants have also been successfully regenerated from protoplasts (Murata et al., 1987; Sihaehakr and Ducreux, 1987). While most of these studies were aimed at plant regeneration in sweetpotato, the frequencies of regeneration reported are not high. In our laboratory, the best shoot regeneration system had a frequency of less than 1% and required about 5 mo. to develop shoots. We were, however, encouraged by the initial results obtained with the two-step protocol for sweetpotato regeneration using leaf explants (described by Medina, 1991), which was adapted to develop an improved system for regeneration of adventitious plants in sweet- potato. The results are reported in this paper.

1To whom correspondence should be addressed at: Plant Molecular and Cellular Genetics Lab, Milbank Hall, Tuskegee University, Tuskegee, Ala- bama 36088-1641.

MATERIALS AND METHODS

Plant material. The stock plants obtained fl'om USDA Plant Introduction Center (Griffin, GA) were maintained as continuous shoot cultures in vitro on the shoot-multiplication medium, which consisted of Murashige and Skoog (MS) salts (Murashige and Skoog, 1962) with the following additions in nlg'liter ~ (wt/vol): myoinositol 100, sucrose 30 000, thiamine hydrochloride 0.4, L-arginine 100, ascorbic acid 200, pantothenic acid 2, putrescine 20, gibberellic acid (GA:~) 20 (Dodds et al., 1991). All media used in this study were adjusted to pH 5.8 prior to autoclaving using 0.1 N NaOH, solidified with Phytagel (Sigma Chemical Co., St. Louis, MO) (3.5 g'liter-1), and au- toclaved at 121 ° C for 20 rain. Plants were maintained in GA7 vessels (Ma- genta Co., Chicago, IL), wrapped with Parafilm, and subcultured evel 3, 6 to 8 wk. All cultures in this study were incubated in growth chambels at 25 + 3 ° C under 16 h photoperiod (50 gM m z s 1).

Explain preparation and culture. Lamina and petiole pieces (0.5 to 1.0 em) were isolated from in vitro grown plants (3 to 4 wk after subculture.). The basal portion of petioles (1 to 2 ram) were carefully sliced off to remove any remnants of the preexisting axillary meristem. The regeneration protocol con- sisted of two stages. The first-slage medium had MS basal salts, myoinositol (100 tug'liter-I), thiamine hydrochloride (0.4 tug'liter-l), sucrose (30000 rag-liter-i), and 2,4 dichlorophenoxyacetic acid (2,4-D) (0.2 tug'liter 1). In the second-stage medium, 2,4-D was substituted by various cytokinins [zeatin riboside (0.2 tug'liter-I), kinetin (0.2 mg-liter-1), or thidiazuron (0.0-0.2 tug'liter 2)]. The growth regulators were filter sterilized and added to the autoclaved media prior to dispensing (25 ml) into petriplates (100 X 15 ram). Most chemicals including 2,4-D and zeatin riboside were obtained from Sigma, while thidiazuron was obtained fi'om Crescent Chemical (Hauppauge, NY).

Petiole pieces were placed horizontally on the nutrient medium in the first stage and vertically with the base (2 to 4 ram) inserted into the medium in the second stage of culture. Lamina were positioned with the abaxial side in contact with the medium in both stages. The duration of incubation in the first stage was 2 to 4 d until the base of petiole (Fig. l A) or the base of the midvein in the lamina began to swell. The second stage where shoot primordia

65

66 GOSUKONDA ET AL.

FIG. 1. Adventitious regeneration of plants in sweetpotato genotype PI 318846-3 using petiole explants. A, Petiole explants prior to culture (left) and after incubation for 3 d on a medium with 2,4-D showing basal swelling (arrows) (right) (Bar = 2 ram). B, A petiole explant (P) exhibiting adventitious shoot (S) and root (R) emergence from the base when cultured for 20 d on a second- stage medium with thidiazuron (0.2 mg-liter-~). The callus part of the base was cut transversely (Bar - 1 mm). C, A petiole explant (P) showing adventitious shoot (S) development, when cultured for 25 d on a medium with thidiazuron (0.2 mg'liter -1) (Bar = 1 ram). D, Petiole explants showing adventitious shoot and root production when cultured for 28-30 d on a medium with thidiazuron (0.2 mg'liter 1). Petriplate size is 100 × 15 ram. E, Sweetpotato plant regen- erated adventitiously from a petiole explant, grown in soil. (Pot rim diam. = 22.9 cm).

PLANT REGENERATION IN SWEET POTATO

TABLE 1

INFLUENCE OF TYPE OF CYTOKININ (IN THE SECOND STAGE) ON SHOOT REGENERATION FREQUENCIES IN PETIOLE EXPLANTS OF SWEETPOTATO GENOTYPES ",~','

67

Zeatin Riboside Kinetin Thidiazuron Genotypes (0.2 rag- liter-~) (0.2 rag-liter ') (0.2 mg-liter 1)

PI 531143 15.7 _+ 6.6 0 10.6 _+ 6.0 PI 318846-3 29.5 _+ 7.8 20.5 _+ 4,6 78.2 + 7.8 Beauregard 12.7 _+ 4.6 28.0 _+ 8.0 0 Regal 18.0 + 5.0 38.8 + 9.7 0 Jewel 0 0 0 Rojo Blanco 16.7 + 6.6 40.9 + 10 0 Hi Dr), 20.0 _+ 7.6 23.3 _+ 8.3 0

2,4-D (0.2 rag-liter-i) used in first stage of regeneration protocol. explants developing shoots, with standard error.

b Data observed after 28-30 d of incubation. Results expressed as percent of

appear lasted fi'om 2 to 6 wk. At least five explants were included in each replication and each treatnmnt was repeated a minimum of thine times. All experiments were repeated at least twice. Macroscopic observations on the appearance of shoots were made daily, and data collected at regular intervals. Shoot regeneration frequency was computed as the proportion of explants showing shoots multiplied by 100.

Histological procedures. Petiole segments that had been grown in culture for 1, 2, 3, or 4 wk and that exhibited swelling (first week), callas proliferation (second and third week), and adventitious shoot development (fourth week) were pi-epared for histological observations according to the procedures of Kuang et al. (1992). Shm't (3 to 5 ram) segments of petiole explants from both swollen and nonswollen areas were fixed in 3% phosphate buffered glutar- aldehyde (pH 6.8) for 24 h with an initial 30 rain of aspiration at 20 psi. Segments were stored at 4 ° C fi/llowing initial fixation at room temperature. Following fixation, segments were rinsed twice in buffer for a total of 30 rain and then passed through a graded ethyl alcohol series before infiltration and embeddment in glycol methacrylate 0B-4 embedding medium, Polyseiences, Wan'ington, PA). Sections (2 p,m) were cut using glass knives on a JB-4 microtome and stained with periodic aeid-Schiffs reagent and aniline-blue black (Kuang et al., 1992). Sections were photographed with Plus-X pan film on a Nikon Biophot photomicroscope.

RESULTS

Sweetpotato petiole pieces were cultured for 2 to 4 days on 2,4-D (0.2 rag-liter t) until the base of petiole began to swell (Fig. 1 A). They were then transterred to a nutrient nledium containing zeatin riboside, thidiazuron, or kinetin (each at 0.2 tug'liter t). The use of kinetin and zeatin riboside produced regeneration from a broad spec- trum of genotypes including some elite eultivars such as Beauregard, Regal, Rojo Blanco, and Hi Dry but the levels of regeneration fre- quency were low (Table 1). Thidiazuron produced the highest regen- eration frequency among the three cytokinins tested with 78.2% of the petiole explants of PI 318846-3 developing shoots (Table 1). However, with the exception of PI 531143 (10.6% shoot regenera- tion), none of the other five genotypes cultured on thidiazuron de- veloped any shoots but instead developed callus (Table 1). When explants were cultured on a medium without 2,4-D during the first stage and transfen'ed to a TDZ (0.2 mg'liter 1) medium for the sec- ond stage, no shoot regeneration was observed (data not shown).

In regenerable explants, typically, the petiole base started to swell in 2 to 3 d (Fig. 1 A). When transfen'ed to a second-stage medium with thidiazuron (0.2 nag'liter 1), a small amount of callus occasion- ally appeared around the base of petiole followed by the emergence of roots and then shoots (Fig. 1 B and i C). Typically, the regenerative petioles produced a single adventitious shoot (Fig. 1 D). Elongation of internodal regions of shoots was achieved by transfer to the shoot

TABLE 2

EFFECT OF LEAF POSITION (PHYSIOLOGICAL MATURITY OF THE EXPLANT) ON REGENERATION EFFICIENCY OF PETIOLE

EXPLANTS OF SWEETPOTATO 'J'

Leaf Position' Percent Regeneration '~-'

1 75.0 + 13.2 2 86.7 + 8.8 3 85.0 _+ 10.4 4 65.0 + 13.2 5 60.0 _+ 13.2 6 41.6 + 4.4

"In the regeneration protocol, the first stage medium was supplemented with 2,4-D (0.2 mg'liter -1) and the second stage medium with thidiazuron (0.2 rag" liter 1). f'Genotype PI 318846-3. ~Petioles taken from leaf position numbers shown in Fig. 2. dData observed after 28-30 d of in- cubation. ~Results expressed as percent of explants developing shoots, with standard en'or.

elongation medium, which was identical in composition to the shoot multiplication medium.

Petioles taken from leaves at various stages of development (Fig. 2) were compared for shoot regeneration competency in the most regenerable genotype, PI 318846-3 using the two-stage protocol (2,4-D at 0.2 nlg'liter 1 followed by thidiazuron at 0.2 mg-liter-1). Very high shoot regeneration (86.7% and 85.0%) was observed in petioles isolated from nodes #2 and #3 on the shoot (Table 2). Pet- ioles fi'om these nodes were also most rapid in their regeneration response with 65 to 68% of the explants developing shoots within 21 d (data not shown).

Lamina and petiole explants taken from nodes #2 and #3 of the in vitro plants (Fig. 2) were tested for their shoot regeneration ability at different concentrations of thidiazuron (0 to 0.4 tug'liter ~) after their exposure to 2,4-D (0.2 tug'liter -~) for 2-4 d. Petioles always exhibited greater regeneration compared to the lamina, and showed 85% shoot regeneration using thidiazuron at 0.2 mg'liter ~ (Table 3). In contrast, lamina showed a maximum of 25.0% regeneration at thidiazuron, 0.1 and 0.2 mg'liter i (Table 3). Although petiole ex- plants developed adventitious shoots at all levels of thidiazuron tested (Table 3), those cultured on thidiazuron at 0.2 mg'liter showed rapid shoot emergence with 62.5% of the explants producing visible shoot primordia by 15 d (data not shown).

Histological examination of cross sections of petiole segments from sweetpotato seedlings revealed no evidence of axillary bud primor-

68 GOSUKONDA ET AL.

\ ,

FIG. 2. Developmental stages of the leaf in the donor plant of sweetpotato from which explants differing in maturity were taken for tissue culture. Donor plants were in vitro stock plants multiplied through nodal culture.

dium or other preexisting meristematic tissue (Fig. 3 A). The outer- most layer of ground tissue cells directly underneath the epidermis consists of prominent chlorenchyma cells. The remaining ground pa- renchyma cells are highly vacuolated and devoid of chloroplasts. Up to five strands of vascular tissue are embedded within the ground tissue, with the three most prominent strands located near the center of petiole and arching upward slightly towards the dorsal side of the petiole.

After the first week of culture, observations of petiole cross sec- tions from nonswollen regions of the cultured petioles demonstrated that chlorenchyma cells directly underneath the epidermis had undergone one or more cell divisions as revealed by cells that shared prominent cross walls (Fig. 3 B). Frequently, these recently divided cells appeared in files of three or four indicating they had recently divided. Files of recently divided cells also were observed in the outer most regions of the vascular bundles. These meristematic cells were directly adjacent to much larger highly vacuolated ground tissue cells that showed no evidence of cell division activity (Fig, 3 C).

Observations of sections taken from swollen regions of petioles revealed considerably more cell divisions as demonstrated by large numbers of recently divided cells associated with the chlorenchyma tissue layer in the outer ground tissue, files of procambial-like cells associated with the vascular bundles, and substantial numbers of recently divided cells dispersed throughout the ground tissue (Fig.

3 D). The presence of large, vacuolated, nonmeristematic cells in- terspersed between and among meristematic cells of the ground tis- sue including the outermost chlorenchyma layer and recently divided cells of the vascular bundles suggests that differentiation of paren- chyma cells occurred de novo at several different places in the pet- iole. There was no evidence to suggest that resumption of meriste- matic activity of parenchyma cells in the ground tissue of petiole segments was associated with the axillary bud primordium, which would be confined to the basal-most part of petiole segments im- mediately distal to the stem.

Calluslike proliferation of cells appeared from the peripheral ground tissue cells of petiole explants by the second and third week of culture. These masses of relatively undifferentiated ceils appear to arise near the surface of the explants independent of underlying vascular bundles as evidenced by highly vacuolated undivided cells located between these cells and the vascular bundles (Fig. 3 E).

By the fourth week in culture, adventitious shoots and roots were observed emerging from the explants. Initially, it appeared that the roots and shoots were emerging independently of one another. How- ever, sections made through the organs from which adventitious shoots were emerging revealed that these organs were rootlike in their morphology as evidenced by a central core of vascular tissue and a cortex region consisting of files of parenchyma cells containing prom- inent multigrained amyloplasts (Fig. 3 F). Some of the files of pa- renchyma cells in the cortex had separated from one another or col- lapsed, which is an additional anatomical characteristic of roots.

Shoot regeneration was observed on petiole pieces irrespective of the part from which they were isolated (i.e., pieces isolated from basal, middle, or top portions of the petiole showed comparable re- generation potential) (data not shown). Adventitious shoots obtained via thidiazuron culture had shorter internodes and appeared stunted. Further, the roots developed from such shoots were small and exhib- ited slower growth. However, when transferred to a shoot elongation medium, thidiazuron-cuhured shoots rooted easily and showed good growth. The rooted plantlets were transferred to plastic containers with sterile soil mix (peat, perlite, and vermiculite in equal propor- tions) and maintained under high humidity for 2 wk. After 2 wk, humidity was gradually reduced, and the plants were finally trans- ferred to pots containing a sterile soil mix and then moved to green- house. There was no mortality in the transplanted plants and nearly 100 plants developed from adventitious shoots grown in the green- house. These appeared healthy and showed no morphological or phe- notypie abnormalities (Fig. 1 E).

DISCUSSION

While there are many reports on production of sweetpotato plants in vitro, most of these protocols are characterized by low frequency of shoot regeneration, long periods of euhure, and frequent media changes. Lowe et al. (1992) estimated the overall frequency of shoot regeneration in sweetpotato to be only 2 to 20%. Alterations in the basal media, combinations of auxins and cytokinins, and the choice of explant sources and plant genotypes have yielded adventitious regeneration of sweetpotato in the past (Kuo, 1991). In our hands, the levels of shoot regeneration obtained from existing protocols were low, and the results were not consistently reproducible.

In our earlier preliminary studies, pulsing of explants with 2,4-D was critical for shoot regeneration, while naphthalene acetic acid or indole acetic acid resulted in only marginal success (unpublished).

PLANT REGENERATION IN SWEET POTATO 69

FIG. 3. Sections of sweetpotato petioles or petiole explants at time 0 and at designated times after explants were incubated on a medium supplemented with 2,4-D or thidiazuron. A, Cross section of a sweetpotato petiole. A single layer of chlorenchyma cells (arrow) is present directly underneath the epidermis. Five bundles of vascular tissue (arrowheads), three median and two lateral, are present within the ground tissue. X 81. B, Cross section of a petiole explant after the first week of culture. Chlorenchyma cells directly underneath the epidermis have undergone one or two cell divisions as evidenced by two or three files of cells sharing common cross walls (arrows). × 330. C, Cross section of a vascular bundle from a petiole explant after 1 wk in euhure. Recently divided cells are evident in the periphery of the bundle (arrowheads) adjacent to large, vacuolated ground tissue cells (double arrowheads) that do not exhibit any meristematie activity. × 311. D, Cross section of a petiole explant after 1 wk in culture exhibiting considerable meristematic activity both within the ground tissue (arrowhead) and vascular bundles (doable arrowheads). Note the large, vacuolated cells within the ground tissue (arrows) that have not resumed meristematic aet ivi ty .× 78. E, Cross seetion of a petiole explant after 2 wk in eulture illustrating two regions of callus proliferation (CP). No meristematic activity is evident between these sites and an adjacent vascular bundle (VB). × 31. F, Cross seetion of an adventitious root after 4, wk in culture. An adventitious shoot (AS) has emerged from this root and shows vaseular connections with the vascular core (VC) of the root. Note the large cortical cells containing amyloplasts (arrowheads) and the large cortical air spaces (double arrowheads). These latter two features are commonly observed in the cortex of roots. × 75.

70 GOSUKONDA ET AL.

TABLE 3

EFFECT OF LEVELS OF THIDIAZURON DURING THE SECOND STAGE ON REGENERATION FREQUENCY IN PETIOLE AND LAMINA

EXPLANTS OF SWEETPOTATO GENOTYPE PI 318846-3 ''b'"

Levels of TDZ (rag-liter-') Petiole Lamina

0.0 19.8 ± 3.7 0 0.1 66.7 --+ 6.4 25.0 ± 7.2 0.2 85.0 ± 7.6 25.0 ± 8.6 0.3 65.0 ± 8.7 10.0 ± 2.9 0.4 36.5 ± 6.9 4.5 ± 2.6

"Results expressed as percent of explants developung shoots, with stan- dard error. ~'Genotype used was PI 318846-3. "Data observed after 28-30 d of incubation.

An appropriate concentration of 2,4-D (0.2 mg'liter 1) and the du- ration of exposure to this chemical during the first stage was impor- tant. When shoot prinmrdia emerged, very little callus was observed at the base of petiole explants (Fig. 1 C and 1 B). Our studies with petiole explants show similar results to that of Medina (1991) where adventitious shoots were formed on leaf explants of Peruvian sweet- potato lines and cv. Jewel cultured on 2,4-D at 0.2 mg-liter ~ in the first stage medium and zeatin riboside (0.2 mg'liter 1) in the second stage.

Although adventitious shoot regeneration from petiole explants of sweetpotato has been reported earlier (Hwang et al., 1983; Xin and Zhang, 1987; Suga and Irikura, 1988), it has been acknowledged that this explant is generally recalcitrant to shoot regeneration (Dodds et al., 1992). Because the petiole is the most suitable organ for Agrobacterium-mediated gene transfer in sweetpotato (Blay et al., 1992; Prakash and Varadarajan, 1992), it is desirable to de- velop a shoot regeneration system from this explant.

The adventitious shoot regeneration observed in our study repre- sents a de novo induced meristem rather than a proliferation of a preexisting meristem. Shoot regeneration was achieved from all parts of the petiole including those sections closer to the lamina where the likelihood of preexisting meristems is nonexistent. In addition, the adventitious shoot regeneration was highly genotype-specific and was achievable only with the use of TDZ. From our experience with micropropagation of sweetpotato, the proliferation of preexisting mer- istems is fairly nonspecific in terms of genotype and can be easily achieved with less-potent cytokinins such as BAP and kinetin. Fi- nally, meristematic activity as evidenced by the proliferation of a large number of cells occurred within the cells of the vascular bun- dles as well as throughout the ground tissue. Pockets of highly vac- uolated parenchyma cells were interspersed among these meriste- matic regions of the ground tissues, further suggesting that dedifferentiation of parenchyma cells in the ground tissue occurred independently of that within the vascular bundles. The fact that ad- ventitious shoots were observed emerging from adventitious roots is consistent with the observation that roots emerged before shoots (Fig. 1 B and 1 C). Further histological studies will be necessary to de- termine if organ regeneration always follows that sequence.

When cut petioles were tested for their shoot regeneration effi- ciency using zeatin riboside, kinetin, or thidiazuron in the second stage, the highest regeneration was observed with thidiazuron but primarily in the genotype PI 318846-3 (Table 1). Thus, thidiazuron- mediated shoot regeneration appears to be genotype specific, while such a specificity was less pronounced with zeatin riboside or ki-

netin. Thidiazuron (N-Phenyl-N'-l,2,3-thidiazol-5-yl urea; Dropp TM) is a derivative of phenylurea and has been reported to possess a strong cytokinintike activity in a number of crops (Mok et al., 1987; Huetteman and Preece, 1993). It has been used successfully to in- duce shoot regeneration in diverse plant species including woody plants such as apple (Sriskandarajah et al., 1990) and eastern cot- tonwood (Prakash and Thielges, 1989), and in leguminous plants such as beans and peanut (Gill and Saxena, 1992; Malik and Saxena,

1992; Matand et al., 1994). The choice of genotype is the most important factor to consider

when regenerating plants in vitro (Ritchie and Hodges, 1993). While our results show considerable specificity of sweetpotato genotypes in their regenerating ability, such specificity is not uncommon in plant tissue culture (Ritchie and Hodges, 1993). Templeton-Somers and Collins (1986) have found significant heritable differences in the morphogenetic responses of sweetpotato genotypes. However, the high shoot regenerability of the genotype PI 318846-3 on the thi- diazuron-supplemented medium has been reproduced nearly 30 times in our laboratory during the past 2 years. This genotype is native to Timor and was imported to the United States from New Zealand (R. Jarret, personal communication). Despite the genotype specificity of thidiazuron treatment, the choice of this chemical used in combination with PI 318846-3 will still be very useful as a model system for sweetpotato tissue culture because of the rapidity and high frequency of shoot regeneration.

ACKNOWLEDGMENTS

Contribution number 226 of the George Washington Carver Agricultural Experiment Station, Tuskegee University. We wish to thank Mr. Fabricio Medina for useful discussions, Mr. Kanyand Matand and Mr. Korsi Dumenyo for technical assistance, Mr. Roger Hagerty tor the drawing of Figure 2, and Ms. Sue Loomis for assistance with photography. We also thank Dr. J. H. M. Henderson and Dr. Robert Locy for their critical review of the manuscript and useful comments and Ms. Cecilia Mosjidis of Auburn University for her assistance with histology and microscopy studies. Research supported by grants from USDA (ALX-9201871) and from NASA (NAGW-2940).

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