Plant regeneration from callus cultures of salt marsh hay, Spartina patens, and its cellular-based salt tolerance

Aquatic botany ELSEVIER

Aquatic Botany51 (1995) 103-113

Plant regeneration from callus cultures of salt marsh hay, Spartina patens, and its cellular-based salt tolerance Xianggan Li, Denise M. Seliskar, Jennifer A. Moga, John L. Gallagher* Halophyte Biotechnology Center, Collegeof Marine Studies, Universityof Delaware, 700 PilottownRoad, Lewes, DE 19958, USA Accepted 11 January 1995

Abstract Salt marsh hay, Spartina patens (Ait.) Muhl. (Poaceae), is a perennial salt-tolerant grass common in salt marshes and sand dunes of the Atlantic and Gulf coasts of the USA, and grows vigorously at coastal seawater salinity. To study the salt tolerance mechanisms that operate in S. patens at the cellular level, a tissue culture and regeneration protocol for this species was developed. Callus was initiated from seedling mesocotyl on ADM medium (Murashige and Skoog (MS) salts + 3% sucrose + 1 mg 1-I indoleacetic acid (IAA) and 1 mg 1-1 2,4-dichlorophenoxyacetic acid (2,4-D)). Regenerable callus was selected from the several morphotypes that developed and was maintained on BND medium (MS salts + 3% sucrose + 0.5 mg 1-1 6-benzylaminopurine(BAP), 1 mg 1-J 1naphthaleneacetic acid (NAA), 0.5 mg 1-1 2,4-D, and 50 ml 1-1 coconut water (CW)). Shoots formed from 90% of the cultures grown on shoot regeneration medium containing BAP and IAA. Roots formed from shoots when they were transferred to root regeneration medium containingindole3-butyric acid (IBA) and activated charcoal or reduced strength MS medium. Plants regenerated via organogenesis have flowered and set viable seeds in a saltwater-irrigated field plot. Dry weight accumulationof unadapted callus at 510 mM NaC1 is similar to that at 0 mM NaC1 (control), indicating that S. patens has strong salt tolerance at the cellular level. Keywords: Spartinapatens; Halophyte; Salt tolerance;Callusculture;Regeneration

1. Introduction Spartinapatens (Ait.) Muhl. (Poaceae) is an important component of salt marsh communities, along the Atlantic and Gulf coasts of the USA. Whole plants of this halophyte * Correspondingauthor. Telephone:(302)-645-4264.Fax: (302)-645-4028. 0304-3770/95/$09.50 © 1995 ElsevierScienceB.V. All rights reserved SSD[0304-3770(95)00454-8

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species are highly salt-tolerant, displaying several salt tolerance mechanisms, such as salt glands and exclusion at the root surface. For example, Pezeshki et al. (1987) reported that salt excretion from leaf surfaces of S. patens occurred within 3-5 days after the addition of salt to the growth media, and stomatal conductance and net photosynthesis of whole plants were affected as soil salinity increased (Pezeshki, 1991). However, the degree of cellularbased salt tolerance of S. patens has not been studied, owing to the lack of a tissue culture system for this species. The purpose of our research was to develop a tissue culture and regeneration procedure to be used in the investigation of S. patens salt tolerance at the cellular level. With the ability to regenerate roots and shoots independently of one another on various differentiation media, these techniques also make possible the future study of the relationships of salt tolerance mechanisms based in the roots and shoots. Further, reliable tissue culture protocols provide opportunities for investigation of plant responses at the molecular level.

2. Materials and methods

Mature seeds collected from natural salt marshes in Delaware were stored at 4°C, either dry or in half-strength seawater. Caryopses of Spartina patens, with intact lemma and palea, were removed from their glumes and disinfected by shaking in a solution of 20% commercial bleach (5.75% sodium hypochlorite by weight), 10% sterile ethanol, plus 0.1% Tween-80 surfactant for 15 min, followed by three rinses of sterile water. Caryopses were then plated on a solidified medium containing Murashige and Skoog (NIS) salts plus 3% sucrose for germination. After 5 days, sterile seedlings were screened and transferred onto 25 ml of callus induction (CI) medium (Table 1). MS revised salt medium containing 100 mg 1-~ inositol and 0.1 mg 1-~ thiamine-HC1 was used throughout most of this study (Murashige and Skoog, 1962). All media were adjusted to pH 5.7 prior to autoclaving at 18 psi for 20 min. Gel-Gro, 0.2% (ICN Biomedicals, Costa Mesa, CA) was used to solidify the media. Cultures were grown in 25 m m x 150 mm tubes or Petri dishes under 30/zE m 2 s - ~ light provided by fluorescent bulbs. After culture on CI medium, callus was maintained on BND medium (MS salts + 3% sucrose + 0.5 mg 11 BAP, 1 mg 14 NAA, 0.5 mg 14 2,4-D, and 50 ml 14 CW), and subsequently transferred onto shoot regeneration (SR) medium (Table 1). Then shoots were transferred onto root regeneration (RR) medium (Table 1). Regenerated plants were separated and acclimated to soil in the humid environment of a jar covered with plastic wrap. Later, plants were potted in the soil in the greenhouse, then moved into a field plot irrigated with coastal seawater. Regenerable callus was investigated by light microscopy using standard histological techniques (Johannsen, 1940; Seliskar, 1985) and by electron microscopy using standard methods (Harris, 1991). Salinity treatment of the callus was with BND medium supplemented with 0 (control), 170, 340, and 510 mM NaC1 for 36 days without a gradual stepwise increase in salinity.

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Table 1 Componentsof media used in cultureand regenerationof Spartina patens Code

MS (g) Sucrose (g) Auxin

CI

SR

RR

ADM ADCC MS2D BND

4.6 4.6 4.6 4.6

30 30 30 30

IB IBI 3B 3BI

4.6 4.6 4.6 4.6

30 30 30 30

BND

BND BND1 BND2 BND3

4.6 4.6 4.6 4.6

30 30 30 30

MS

1/2MS I/4MS

2.3 1.2

15 7.5

IK/IKC

5IK 10IK 5IKC 10IKC

4.6 4.6 4.6 4.6

30 30 30 30

B/BI

1 I; 1 D 1 I; 1 D 2D 1 N; 0.5 D

BAPor kinetin

50 0.5 B

0.21

1B 1B 3B 3B

1 N; 0.5 D 1 N; 0.1 D 1N 0.5 N

0.5 B 0.5 B 1B 1B

5 Ib 10 Ib 5 Ib 10 Ib

0.1 K 0.1 K 0.1 K 0.1 K

0.21

CW (ml) Charcoal(g)

5

50

50

50 50

Concentrations are in mg 1-t for hormones and others are gram or ml 1-1. CI, Callus induction; SR, shoot regeneration;RR, root regeneration;MS, Murashige and Skoog salts; CW, coconutwater; D, 2,4-D; I, IAA; Ib, IBA; N, NAA;B, BAP; K, kinetin.

3. Results and discussion 3.1. Callus induction and maintenance Spartina patens caryopses showed slightly less contamination when disinfected in 20% bleach plus 10% ethanol for 15 min than in 10% bleach plus 10% ethanol for 20 min. A severe reduction in germination occurred when the higher concentration of bleach was combined with a longer time for sterilization. The addition of 10% ethanol reduces air bubbles which form close to the seed surface, thereby increasing the effectiveness of sterilization. We reduced cross-contamination by separating seeds on MS media (supplemented with 3% sucrose and 0.2% Gel-Gro in Petri dishes) until they germinated (5 days). After germination, those seedlings contaminated by bacteria were eliminated and the sterile seedlings moved to callus induction medium. The most promising medium for S. patens callus formation from seedlings (Plate 1 ( 1) ) was the A D M medium (MS salts + 3% sucrose, + 1 mg 1-1 indoleacetic acid (IAA) and 1 mg 1-1 2,4-dichlorophenoxyacetic acid ( 2 , 4 - D ) ) . Coconut water (CW) is thought to contain cytokinins, which would explain the reduced callus formation that resulted when C W was added to the A D M medium, as previous research has shown that a combination of high auxin and low cytokinin concentration induced callus formation in the Poaceae (Evans et al., 1981). Activated charcoal was added to cultures of S. patens to enhance callus

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Plate 1. Callus induction and maintenance. ( 1 ) Callus initiating on seedling cultured on ADM medium. C, Callus; R, root. Bar represents 600 p,m. (2) Tissue culture initiating on germinated seed cultured on BND medium. C, Callus; S, shoot; R, root. Bar represents 400 p.m. (3) Inflorescence after approximately 2 months in culture. Gc, Green callus; Wc, white callus. Bar represents 600 p.m. (4) Development of green spots into organized areas on BND medium. Gs, Green spots; Ws, white spots. Bar represents 200 p,m. (5) Excised callus tissue. Ne, Nonembryogenic white callus; Gc, green compact callus. Bar represents 600 p,m.

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formation and prevent the formation of a mucus coating on the callus. BND medium is also useful in callus formation (Plate 1 ( 2 ) ) . The green callus produced on BND exhibits vigorous proliferation and regeneration. All four callus induction media produced viable calli. Callus was formed at the juncture of shoot and mesocotyl or root and mesocotyl after being cultured for about 2 weeks. The mesocotyl of S. patens elongated and swelled. The BND medium is better than ADM or MS2D (MS salts + 3% sucrose + 2 mg 11 2,4-D) for maintenance of S. patens callus cultures. The average diameter of callus after 2 weeks of culture was 0.52 cm on BND, 0.46 cm on ADM, and 0.41 cm on MS2D. The callus on BND medium was primarily green mixed with some white and a little brown (Plate 1 (4)). Callus on ADM medium was brown (Plate 1 (1) ) whereas that on MS2D was yellow. The compact green callus cultures grew best on BND medium (Plate 1 (5)) and were therefore exclusively maintained on this medium in subsequent subcultures. The yellow callus either on MS2D or MS1D (the same as MS2D but with 1 mg 1-~ 2,4-D) gradually formed compact golden callus, which produced less mucus when cultured on Whatman (Hillsboro, OR) No. 1 filter paper on the surface of the medium than when cultured directly on the medium. Bright white callus was formed from inflorescence explants on B5D2S 10 medium (B5 salts (Gamborg et al., 1968) + 2 mg 1- ~ 2,4-D + 10% sucrose), and green mixed with white calli (Plate 1 (3)) were observed on the B5D2K0.5S3 medium (B5 salts + 2 mg 2,4D and 0.5 mg kinetin 1-1 with 3% sucrose). 3.2. Shoot and root regeneration

Shoot regeneration was accomplished by transferring green callus onto B or BI media. The removal of auxin in all cases caused the tissue to become dark green (Plate 2 ( 1 ) ). Thirty to forty tiny shoots in 1 cm diameter of callus were visible after the callus was moved from BND to both B (MS salts + 3% sucrose + 1 mg 11 BAP) and BI media (same as B, but + 0.2 mg 1-11AA) for 2 weeks (Plate 2 (2,3)) and Plate 3 (1)). After 1 month of culture, regenerated shoots reached up to 5 cm in height. The MS medium without hormones was the least efficient for shoot formation. The regenerated shoots per tube on various SR media are shown in Fig. 1. The highest and most consistent yield of shoots was obtained by transferring callus from BND onto 3B (MS salts + 3% sucrose + 3 mg 1"~ BAP) or 3BI (same as 3B, but with 0.2 mg 1-~ IAA) (Fig. 1). Shoots did not spontaneously root on the SR medium. Before transferring to root regeneration medium, the amount of auxin in the BND medium was slowly decreased by subculturing on the following sequential media: BND, BND1, BND2, and BND3, every 2 weeks. Similar numbers of shoots were formed without any rooting. Root regeneration was achieved by transferring larger shoots to RR media. Rooting shoots per tube sample were pooled and the percentage is shown in Fig. 2. Rooting occurred within 1 month on both IKC (MS salts + 3% sucrose + 5 mg 1-11BA + 0.1 mg 1-1 kinetin + 50 g 11 charcoal) and MS media. A striking decrease in rooting was found on IK media (MS salts + 3% sucrose + 5 mg 1-1 IBA + 0.1 mg 11 kinetin) without charcoal (Fig. 2) compared with IK with charcoal (IKC). Reduced-strength MS medium for rooting was better than IK and almost as good as IKC. Attempts to simplify the regeneration protocol were successful, and are summarized in a flow chart (Fig. 3). Induced callus from ADM

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Plate 2. Shoot meristem and shoot regeneration. ( 1) Shoot meristems ( SM ). Bar represents 220/~m. (2) Multiple shoots (MS) from meristems. Bar represents 150/.~m. (3) Green plants (GP) regenerated from cultures. Bar represents 300/~m. (4) Regenerated whole plants in flower in the greenhouse. S, Seed-containing inflorescences. Bar represents 7.5 cm.

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Plate 3. Cross-sectionof meristem and shoot regeneration. ( 1) Cross-sectionof shoot regenerationfrom shoot meristems. Ms, Multipleshoots. Bar represents 60 bLm.(2) Close-upof cross-sectionof shoot regenerationfrom shoot meristems. S, Shoot; Oa, organizedarea; La, loose area. Bar represents 30/zm. (3) Transmissionelectron microscopesectionof the cells in the organizedarea. Mt, Mitochondria;Vc, vacuoles.Bar represents 1.5/~m. (4) Transmissionelectronmicroscope section of the cells in the loose area. Vc, Vacuoles. Bar represents 2.5/xm. medium was maintained on BND media, after which only the green callus was transferred to 3B or 3BI medium (shoot regeneration media) for about 1 month. Next, shoots were transferred to the IKC or reduced-strength MS media for root regeneration. Regenerated plants were transplanted first to vermiculite and then to potting soil in the greenhouse. The plants flowered and set viable seeds in the greenhouse (Plate 2 ( 4 ) ) and in a field plot where they were irrigated with coastal seawater. The degree of somaclonal variation in these

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o

o


tD_

E (3 O~

0 0 c-

K.X K)< K)< K)< ( X <,p( <.)<
1B 1BI

3B

3BI

BND

Fig. 1. Shoot regeneration on SR media in Table 1. Each bar represents the mean of 16 samples + SD.

100

80 1

0 0

E o

60

40 c 0 0

20

IK

IKC

MS

Fig. 2. Root regeneration on RR media in Table 1. Each bar represents the mean of 16 samples + SD.

regenerated plants is unknown. Albino plants have not been observed in this culture, although this phenomenon is common in other regenerated halophytic grasses (Distichlis spicata (L.) Greene and Sporobolus virginicus Kunth; Straub et al. ( 1989, 1992) ).

3.3. Histology Histological studies of the callus exhibiting shoot primordia (Plate 2 ( 1) ) on BND media were conducted. Somatic embryogenesis and the torpedo-like structures leading to zygotic

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[

CM

sR

RR

Seedlings

111

]

BND

50 D

B/S: D 3O

IKC

MS

6O D

46 D

Fig. 3. Flow chart of medium sequence used in regeneration. Medium designations are noted in Table 1. D is number of days on media for callus induction (CI), callus maintenance (CM), shoot regeneration (SR), and root regeneration (RR).

embryos were not observed. This fact, together with the regeneration of some complete shoots without roots, provided evidence that organogenesis had been the mode of regeneration. Histological sections clearly demonstrate organogenesis in Plate 3 ( 1), where multiple shoots are evident arising from the organized areas of callus. The shoots may have originated via a de novo organization of meristems (Plate 3 (2)), subsequently developing a group of shoot primordia (Plate 2 (1) ). Leaf primordia emergence followed the development of shoot primordia (Plate 3 ( 1) and Plate 2 ( 1) ). Centralized xylem development and polarized cell growth were observed around the organized area (Plate 3 (1)). Dissection of callus cultures showed organized areas of compact cell growth with small, isodiametric, typical embryogenic type cells (Plate 3 (2)), as well as areas of looser cell growth with typical nonembryogenic cells that are larger and oval (Plate 3 (2)). Plate 3 (3) is a transmission electron micrograph of S. patens callus showing an isodiametric embryogenic cell. Such embryogenic cells have been characterized in the literature as small, isodiametric cells with prominent nuclei, dark staining cytoplasm, small vacuoles, and large accumulations of starch granules. The cells in the loose area are highly vacuolated with little accumulated starch and exhibit cell lysis (Plate 3 (4)). Similar cells have been reported for other species by Heyser and Nabors (1982) and Vasil and Vasil (1984). 3.4. Salt tolerance of callus Results of callus growth under salt stress are shown in Table 2. The fresh weight accumulation of calli grown with 170, 340, and 510 mM NaCI was 91%, 74%, and 67% of the control, respectively. Dry weight accumulation of callus was 106%, 102%, and 95% of control, respectively; however, these differences do not represent a significant change in response to salt. The changes in ash-free dry weight (AFDW) and ash content are opposite and offset each other. AFDW was 99%, 93%, and 86% of control, respectively, at the three

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Table 2 Callus grownon BND mediasupplementedwith increasingamountsof NaCI NaCl (mM) FW (g)

DW (g)

0 170 340 510

0.2565:0.041 0.2295:0.035 0.271+0.020 0.2265:0.024 0.2625:0.013 0.2145:0.015 0.2435:0.016 0.1975:0.008

1.59+0.26 1.44+0.15 1.185:0.13 1.06+0.07

AFDW(g)

WC (%)

AC (%)

86.85:2.3 81.15:1.2 77.75:2.9 77.05:0.9

10.5+0.5 16.85:3.2 18.35:2.6 18.7+2.6

Callus, with the initial amount of 0.5 g (FW), was cultured on the media for 36 days. Water content (WC) = ( F W - D W ) / F W , where FW is fresh weight and DW is dry weight; ash content (AC) = (DW- AFDW)/DW, where AFDW is ash-free dry weight. Numbers are the means of four replicates 5:SD. salinities. Concurrent with the slight decline was a decrease in water content from 87% to 77% (Table 2). A similar reduction in cellular hydration was seen by Blits et al. (1993) in the salt marsh dicot, Kosteletzkya virginica Presl ex A. Gray. The ash content of S. patens nearly doubled (from 10% to 19%), indicating an accumulation of salt in the cells that continued to grow vigorously. All of these characteristics indicate that S. patens has a high cellular-based salt tolerance. The salt tolerance of the callus of S. patens appears to be greater than that of other halophytes reported in the literature. Fifty per cent growth inhibition was reported to occur at 170 mM NaC1 in callus of Suaeda maritima (L.) Dum. (Von Hedenstrom and Breckle, 1974). Calli of the halophytes Atriplex undulata D. Dietr. and Suaeda australis (R.Br.) Moq. were considered salt-sensitive by Smith and McComb ( 1981). Cell suspension cultures of the salt marsh dicot Kosteletzkya virginica are inhibited in growth by 50% at approximately 230 mM NaC1 (Blits et al., 1993). Cell suspension cultures of the halophytic grasses Distichlis spicata and Spartina pectinata Link were more salt-tolerant than any of the dicots cited, but less salt-tolerant than the callus of S. patens, with 50% growth inhibition occurring at 320 mM and 280 mM NaC1, respectively (Warren and Gould, 1982; Warren et al., 1985).

Acknowledgments Support for this research came from the University of Delaware Sea Grant College Program under Grant NA83AA-D-00017, Project R/B-22 from the Office of Sea Grant, National Oceanic and Atmospheric Administration (NOAA), US Department of Commerce, and from the Coastal Ocean Program Office of NOAA through Grant NA90AA-DSG457 to the University of Delaware Sea Grant College Program, and from the NSF Summer Student Program (for the support of J. Moga). Additionally, we thank Dr. G.L. Zhou and Jinglan Wu for preparing the histological slides, and Dr. J.D. Rao for discussion.

References Blits, K.C., Cook, D.A. and Gallagher, J.L, 1993. Salt tolerance in cell suspension cultures of the balophyte Kosteletzkya virginica.J. Exp. Bot. 44: 681-686.

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Evans, D.A., Sharp, W.R. and Flick, C.E., 1981. Behavior of cell cultures: embryogenesis and organogenesis. In: T.A. Thorpe (Editor), Plant Tissue Culture: Methods and Application in Agriculture. Academic Press, New York. Gamborg, O.L., Miller, R.A. and Ojima, K., 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res., 50: 151-158. Harris, J.R., 1991. Electron Microscopy in Biology: a Practical Approach. Oxford University Press, Oxford, pp. 1-27. Heyser, J.W. and Nabors, M.W., 1982. Regeneration of proso millet from embryogenic calli derived from various plant parts. Crop Sci., 22: 1070-1074. Johannsen, D.A., 1940. Plant Microtechnique. McGraw-Hill, New York, 523 pp. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant., 15: 473-497. Pezeshki, S.R., 1991. Population differentiation in Spartina patens: gas exchange response to salinity. Mar. Ecol. Prog. Ser., 72: 125-130. Pezeshki, S.R., Delaune, R.D. and Patrick, Jr., W.H., 1987. Response of Spartina patens to increasing levels of salinity in rapidly subsiding marshes of the Mississippi River Deltaic Plain. Estuarine Coastal Shelf Sci., 2,*: 389-399. Seliskar, D.M., 1985. Effect of reciprocal transplanting between extremes of plant zones on morphometric plasticity of five plant species in an Oregon salt marsh. Can. J. Bot., 63: 2254-2262. Smith, M.K. and McComh, J.A. 1981. Effect of NaCI on the growth of whole plants and their corresponding callus cultures. Aust. J. Plant Physiol., 8: 267-275. Straub, P.F., Decker, D.M. and Gallagher, J.L., 1989. Tissue culture and regeneration of Distichlis spicata (Gramineae). Am. J. Bot., 76: 1448-1451. Straub, P.F., Decker, D.M. and Gailagher, J.L., 1992. Characterization of tissue culture initiation and plant regeneration in Sporobolus virginicus. Am. J. Bot., 79:1119-1125. Vasil, V. and Vasil, I.K., 1984. Induction and maintenance of embryogenic callus cultures of Gramineae. In: l.K. Vasil (Editor), Cell Cultures and Somatic Cell Genetics of Plants, Vol. 1. Academic Press, New York, pp. 36-42. Von Hedenstrom, H. and Breckle, S. 1974. Obligate halophytes? A test with tissue culture methods. Z. Pflanzenphysiol., 74: 183-185. Warren, R,S. and Gould, A.R., 1982. Salt tolerance expressed as a cellular trait in suspension cultures developed from the halophytic grass Distichlis spicata. Z. Pflanzenphysiol., 107: 347-356. Warren, R,S., Baird, L.M. and Thompson, A.K., 1985. Salt tolerance in cultured cells of Spartina pectinata. Plmlt Cell Rep., 4: 84-87.