PlantCeU Reports

Plant Cell Reports (1991) 10:453-456

9 Springer-Verlag1991

Plant regeneration via somatic embryogenesis in creeping bentgrass (Agrostis palustris Huds.) Heng Zhong, C. Srinivasan, and Mariam B. Sticklen Departments of Crop and Soil Sciences and Entomology, 202 Pesticide Research Center, Michigan State University, East Lansing, MI 48824-1311, USA Received May 23, 1991/Revised version received July 18, 1991 - Communicated by J.K. Vasil

ABSTRACT We have established a high-frequency plant regeneration system via somatic embryogenesis from mature seeds of creeping bentgrass (Agrostis palustris Huds). The effects of 2,4dichlorophenoxyacetic acid (2,4-D), 3.6-dichloroo-anisic acid (dicamba) and 6-benzyladenine (BA) on callus formation and emhryogenesis were evaluated. Callus produced on the Murashige and Skoog (MS) (1962) medium containing 2,4-D had low embryogenic potency. In the presence of 30 laM dicamba, addition of 2.25 to 9 ~M BA significantly enhanced embryogenic callus formation over dicamba alone. Optimum frequency of somatic embryogenesis was achieved on MS basal medium containing 30 laM dicamba and 2.25 laM BA. Over 80% of somatic embryos germinated and formed plantlets on half-strength MS basal medium. These plantlets grew normally in the greenhouse. A b b r e v i a t i o n s : MS = Murashige and Skoog medium; 2,4-D = 2,4-dichlorophenoxyacetic acid; BA = 6benzyladenine; dicamba = 3, 6-dichloro-o-anisic acid.

INTRODUCTION Somatic embryogenesis has been reported for several grass species, e.g. Panicum maximum (Lu and Vasil 1981), Pennisetum americanum ( Vasil and Vasil 1981), Dactylis glomerata (Trigiano et al. 1989), and Cynodon dactylon (Ahn et al. 1985). Of the economically important turfgrasses, bentgmss (Agrostis spp.) is commonly used on golf courses (Beard 1982). Plant regeneration from in vitro cultures of creeping bentgrass (A. palustris Huds.) has been demonstrated Offprint requests to. M.B. Sticklen

previously via organogenesis from seed-derived calli (Blanche et al. 1986; Krans et al. 1981). Here we report high-frequency plant regeneration via somatic embryogenesis in creeping bentgrass. MATERIALS AND METHODS Embryogenic callus formation, embryogenesis, and plant regeneration. Mature seeds (caryopses) of 'Penncross' creeping bentgrass ( 90% viability) were surface sterilized first in 50% ethanol for 5 min, and then in 40% commercial bleach (containing 5.25% sodium hypochlorite) plus 0.1% Tween 20 for 15 min tinder vacuum. Surface sterilized caryopses were rinsed three times with sterile distilled water and cultured in Petri dishes containing 20 ml of MS (Murashige and Skoog 1962) basal medium supplemented with 500 mg/l enzymatic casein hydrolysate (Sigma), 3% sucrose, and different concentrations of 2, 4-D (2.25, 4.5, 9 or 18 pM), dicamba (5, 10, 30 or 60 pM), or combinations of 30 pM dicamba and BA (2.25, 4.5, 9.0 or 18.0 vM). All media were adjusted to pH 5.8 prior to the addition of 7 g/l phytagar (Gibco/BRL) and autoclaved at 121~ C for 20 rain. All cultures were incubated at 24 +__2 ~ C in a culture room under dark, and subcultured every eight weeks. Somatic embryos were transferred to Petri dishes containing 15 ml of half-strength MS basal medium. The cultures were incubated for three weeks tinder continuous fluorescent light (60 laE m "2 s-l). Plants were transferred to clay pots containing a l:l mixture of sand : Bacto Pro Plant Mix (Michigan Peat Co.) and maintained in a greenhouse (800 pE m "2 s "l, at 30 ~ C, 16 h/day). Data collection and statistical analysis. There were five Petri dishes for each growth regulator treatment, each dish containing 200 caryopses. Number of callus clumps (1-2 mm) and number of embryogenic calli, identified as opaque, yellowish, compact and organized (Lu and Vasil

454 1981), were scored from each Petri dish 7 weeks after initial culture. The frequency of callus formation was expressed as the percent of callus clumps produced per total number of caryopses in a Petri dish. The frequency of embryogenic callus formation was expressed as the percent of embryogenic calli produced per total number of calli in each Petri dish. Since most of the primary somatic embryos proliferated secondary and tertiary somatic embryos, it was difficult to accurately quantitate the number of somatic embryos produced from each embryogenic callus. Therefore,the relative efficiency of somatic embryogenesis per embryogenic callus clump was rated visually using all the embryogenic calli and scored as "+++" for those calli producing more than 50 embryos, "++" for those producing 10-50 somatic embryos, "+" for callus with less than 10 embryos and "-" for callus with no somatic embryos. Prior to statistical analysis, all percentage data were transformed using arcsin (Robert and James 1980). Analysis of variance was performed using the software PROC GLM in SAS (Sas Institute, Car),, N. C.). Means were compared at the 1% level using Duncan's Multiple Range Test.

RESULTS The number of caryopses forming callus, the frequency of embryogenic callus formation, and the mean number of somatic embryos per callus varied significantly with the kind and concentration of growth regulators used (Table I). The time interval between the various steps in the regeneration of creeping bentgrass is shown in Table II. The comparative morphology of the calli and somatic embryos is described below. All 2,4-D concentrations induced callus formation. The frequency of callus formation did not vary significantly between media containing 2.25 to 9.0 /aM 2,4-D, but the frequency of callus formation was reduced when caryopses were cultured on medium containing 18/aM 2,4-D. On medium containing 2.25 or 4.5 /aM 2,4-D, caryopses first germinated and small translucent calli proliferated either from root or shoot tissues of the seedlings within the first week of culture. Seed germination was inhibited on the medium containing 9.0 or 18.0/aM 2,4-D, and mainly nonembryogenic calli emerged directly from caryopses. After four weeks of culture on medium containing 9.0 /aM 2,4-D, a few pockets of opaque, yellowish, organized structures appeared, which later developed into somatic embryos. The somatic embryos appeared either singly or in small groups on the surface of the calli. These somatic embryos developed normal green plantlets two weeks after transfer to half-strength MS basal medium and incubatio~ under light.

The frequency of callus formation varied significantly between caryopses cultured on media containing 5 and 10 /aM dicamba. Calli formed on medium containing 30 or 60 /aM dicamba were compact and mucilaginous, and produced only roots after transfer to half-strength MS basal medium. Within three weeks after culture initiation, compact and granulate embryogenic calli were initiated from caryopses on the media containing 30/aM dicamba plus BA. An optimum frequency of callus production was found on medium supplemented with 2.25 or 4.5 /aM BA plus 30 /aM dicamba. At a higher concentration (9.0 or 18 /aM) of BA, callus formation was low (Table I). Somatic embryos were organized in groups from embryogenic callus (Fig. 1). Embryogenic callus formation was significantly high on 9.0 /aM 2,4-D compared to other concentrations. The frequency of embryogenic callus formation increased from 57% on 5/aM dicamba to 79% on the medium containing 10 laM dicamba. Further increase in dicamba concentrations in the medium to 30 or 60/aM drastically reduced the embryogenic potency of the callus (Table I). The addition of BA to medium containing 30/aM dicamba effectively counteracted the inhibitory effect of 30/aM dicamba on somatic embryogenesis. An optimum frequency of embryogenic callus formation was achieved on MS basal medium containing 30 /aM dicamba plus 2.25/aM BA (Fig. 1). The embryogenic calli retained their capacity to efficiently produce somatic embryos even after eight months of maintenance on media containing 30 /aM dicamba and different concentrations of BA. Groups of somatic embryos were easily separated from the callus and germinated precociously when exposed to light (Fig. 2). Over 80% of the somatic embryos produced on media containing 30 laM dicamba and any concentration of BA, formed plantlets three weeks after their transfer to half-strength MS basal medium and on exposure to light. Upon transplantation to a soil mix, about 95% of these plantlets survived the greenhouse acclimation and produced healthy plants [Fig. 3]. DISCUSSION Several genera of grasses have been efficiently regenerated through somatic embryogenesis (Vasil, 1987; Hanna et al. 1984; Artunduaga et al. 1989; Cobb et al 1985; Linacero and Vazquez

455 Table L Effect of growth regulator concentrations on the formation of callus, embryogenic callus, and somatic embryo in A. I~lustris Huds. Growth regulator 2,4-D

dicamba

30 }aM dicamba + BA

Conc. (laM)

Callus formation (%) Mean + SE Duncan Grouping

E. callus formation (%) Mean + SE Duncan Grouping

2.25 4.5 9.0 18.0

71.38+ 70.38 + 69.18 + 55.06+

1.70 0.98 4.41 2.31

A A A B

0.0 + 0.0 3.78 + 7.29 20.00 + 5.47 3.20+ 4.38

B B A B

5.0 10.0 30.0* 60.0*

79.06+ 72.20 + 76.20+ 74.98 +

1.13 4.93 0.57 1.17

A B AB AB

56.74+ 6.17 78.90 + 5.53 7.95+ 6.18 3.40 + 5.00

B A C C

++

2.25 4.5 9.0 18.0

80.76 + 80.12 + 75.24 + 56.70+

1.26 1.57 2.95 2.34

A A B C

91.18 + 87.10 + 83.46 + 81.00+

A A A A

+++

2.86 2.53 2.87 1.35

Efficiency of embryogenesis (per 2-5 mm callus)

-t-

+++ + +

+++ +++ ++

* One replication data was missing.

Table H. Time table for an optimum in vitro regeneration of A_. palustris Huds. Culture Condition

Stage

Medium

Efficiency

Embryogenic callus

MMS

24 + 2~

dark

2-3 mm

3

Somatic embryos

MMS*

24 + 2~

dark

"50

5

Plantlets

1/2 St. MS**

24+ 2~

60 laE m2s 1, 16h day

"40

2

Acclimated plants

SBPPM***

30 + 2~

800 ~tE m2s -1, 16h day

-40

1

Greenhouse plants

SBPPM

30 + 2~

800 taE m2s l , 16h day

306 cm 2 (area/plant)

4

weeks in culture

* M M S = M S basal m e d i u m + 30 laM dicamba + 2.25 laM B A + 0.5 g/l enzymatic casein hydrolysate. ** 1/2 St. M S = half-strength M S basal medium. * * * S B P P M = Sand : Bacto Pro Plant M i x (1:1).

1990). Explants of these grasses were induced to form somatic embryos by 2,4-D. However, in creeping bentgrass this auxin is less effective. Dicamba at 20-30 laM is an effective auxin to produce somatic embryos and plantlets in orchard grass (Conger et al. 1983) and in Kentucky bluegrass (McDonnel and Conger 1984) but in creeping bentgrass 30 laM dicamba inhibited embryogenesis. The inhibitory-effect of 30 laM

dicamba was counteracted by BA. The mechanism of this interaction is unknown. Benzyladenine is usually considered inhibitory to somatic embryogenesis, but the combination of BA and 2,4-D promoted embryogenesis in several dicot and monocot species (see reviews by Raghavan 1986 and Bhaskaran and Smith 1990). The combined effects of dicamba and BA on somatic embryogenesis were not reported before.

456

Fig. 1. Proliferation of calli and somatic embryos from a creeping bentgrass caryopsis eight weeks after culture on MS medium containing 30 pM dicamba and 2.25 pM BA. (S = seed; NE = non-embryogenic callus; SE = somatic embryos) Fig.2. Germination of embryos on a half strength MS basal medium. Fig.3. Creeping bentgrass patch derived from somatic embryos.

A c k n o w l e d g m e n t . Heng Zhong is a Rockefeller Foundation Fellow on leave from the Institute of Botany, Academia Sinica, Beijing, China. The authors are grateful to Dr. Joseph M. Vargas and Mr. A. R. Detweiler for the supply of creeping bentgrass seeds and to Dr. Mark G. Bolyard for critical review of this manuscript.

REFERENCES Ahn BJ, Huang FH, King JW (1985) Crop Sci. 25:11071109 Artunduaga JR, Taliaferro CM, Johnson BB (1989) In vitro Cellular & Developmental Biology 25 : 753-756 Bhaskaran S, Smith RId (1990) Crop Sci. 30:1328-1336 Beard JB (1982) Turf management for golf courses Brahms G (ed) Burgerss Publishing Co. Minneapolis, Minnesota Blanche FE, Krans JV, Coats GE (1986) Crop Sci. 26: 1245-1248 Cobb BG, Vanderzee D, Loescher WH, Kennedy RA (1985) Plant Science 40:121-127 Conger BV, Harming GE, Gray DJ, McDaniel JK (1983) Science 221:850-851

Hanna WW, Lu C, Vasil IK (1984) Theor. Appl. Genet. 67:155-159 Krans IV, Henning VT, Torres KC (1981) Crop Sci. 22: 1193-1197 Linacero R, Vazquez AM (1990) Plant Sci.72:253-258 Lu C, Vasil IK (1981) Theor. Appl. Genet. 59:275-280 McDaniel RE, Conger BV (1984) Crop Sci. 24:573-57 Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 Raghavan V (1986) Embryogenesis in Angiosperms - A developmental and Experimental Study. Cambridge Univ. Press, Cambridge pp 303 Robert GDS, James HT (1980) Principles and procedures of statistics. McGraw-Hill Book Co. N.Y. Trigiano RN, Gray DJ, Conger BV, McDaniel JK (1989) Bot. Gaz. 150:72-77 Vasil V, Vasil IK (1981) Am. J. Bot. 68:864-872 Vasil IK (1987) J. Plant Physiol. 128:193-218