Relationship of abscisic acid to somatic embryogenesis in Dactylis glomerata

Environmental and Experimental Botany, Vol. 33, No. 4, pp. 4 9 5 4 9 9 , 1993 Printed in Great Britain.

009~8472193 $6.00 + 0.00 © 1993 Pergamon Press Ltd

R E L A T I O N S H I P OF ABSCISIC ACID TO S O M A T I C EMBRYOGENESIS IN D A C T Y L I S GLO ME R A TA L. M. BELL,* R. N. TRIGIANO* and B. V. CONGER t

*Ornamental Horticulture & Landscape Design and ]'Plant and Soil Science, Institute of Agriculture, University of Tennessee, Knoxville, TN 37901, U.S.A.

(Received 8 February 1993; accepted in revisedform 28 May 1993) BELL L. M., TRIGIANOR. N. and CONGER B. V. Relationship ofabscisic acid to somatic embryogenesis in Dactylis glomerata. ENVIRONMENTALAND EXPERIMENTALBOTANY 33, 495--499, 1993. The influence of abscisic acid (ABA) on somatic embryogenesis in orchardgrass (Dactylis glomerata L.) was studied in one embryogenic (Embryogen-P) and one nonembryogenic (I-39) genotype. Leaf segments from the basal portions of the two innermost leaves were cultured for various lengths of time on SH medium containing 0.01-20.0 #M ABA. Somatic embryogenesis was increased in Embryogen-P by a 3-day exposure to 0.01-10.0/~M ABA; 20 #M was inhibitory. 1-39 did not respond to any ABA treatment. Quantification by enzyme-linked immunosorbent assay (ELISA) indicated that exogenous ABA levels in leaf tissues were nearly the same in the two genotypes at the initiation of culture but decreased much more rapidly during the first 7 days in the nonembryogenic than in the embryogenic genotype. They were the same after 14 days of culture. These results suggest that somatic embryogenesis in the embryogenic genotype may be increased by manipulation of concentration and duration of ABA application. Attempts to induce somatic embryogenesis in the nonembryogenic genotype with ABA treatments were not successful.

Ke)~ words: ELISA, in vitro, orchardgrass, plant growth regulator.

Previous work with orchardgrass showed that zeatin, a n a t u r a l l y occurring cytokinin, a d d e d TIssuE culture techniques for the f o r m a t i o n of exogenously to the m e d i u m in concentrations as low as 10 -9 M suppressed somatic embryogenesis somatic e m b r y o s have been developed for m a n y species. !~3'17) However, the factors controlling this from leaf cultures in an e m b r y o g e n i c genotype./16~ process are still chiefly unknown, a n d most species F u r t h e r m o r e , endogenous levels of zeatin, zeatin in the Poaceae have been p a r t i c u l a r l y difficult to riboside, d i h y d r o z e a t i n and d i h y d r o z e a t i n ribom a n i p u l a t e in vitro. (14) Factors involved in somatic side were three- to four-fold higher in two nonembryogenesis o f Dactylis glomerata L. (orch- e m b r y o g e n i c genotypes than in the e m b r y o g e n i c genotype.~ 16) ardgrass) have been studied in an a t t e m p t to better u n d e r s t a n d this p h e n o m e n o n . These include Abscisic acid (ABA) is a senescence p r o m o t o r genotype, c3) p l a n t g r o w t h regulators/8'16) a n d a n d hastens senescence of d e t a c h e d leaves./9) T h e other m e d i u m supplements./2'~° 12/ m a i n objective of this study was to d e t e r m i n e if ABA, a d d e d to culture m e d i u m , would increase Cytokinins are the most effective p l a n t g r o w t h somatic embryogenesis in an e m b r y o g e n i c genoregulators d e l a y i n g senescence a n d are more effective in d e t a c h e d t h a n in a t t a c h e d leaves./4/ type a n d induce the response in a non495 INTRODUCTION

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embryogenic genotype. A second objective was to relate changes in endogenous ABA levels during culture to the embryogenic response of both genotypes. This was determined by enzyme-linked immunosorbent assay (ELISA).

after 21 days. The number of embryos formed was assessed as the number of plantlets counted divided by the germination rate. /~Ji Differences in the embryogenic response between ABA treatments and their corresponding controls were determined by a one-sided, paired t-test.

MATERIALS AND METHODS

Orchardgrass plants used in tissue culture and for ABA quantification experiments were grown in an environmentally controlled growth chamber on a 16-hr 22°C light 8-hr 15°C dark cycle. Light was provided by fluorescent tubes and incandescent bulbs at an intensity of 100 #mol m ~ s ~. Embryogen-P, '1) a genotype that produces somatic embryos when cultured in vitro (embryogenic), and one nonembryogenic genotype, 1-39~ were used in all experiments. L Tissue culture The basal 3 cm of the two innermost leaves was split along the midvein to form "sister halves". These were surface sterilized by stirring for 2 min in 2.6% sodium hypochlorite containing approx. 0.1% v/v Triton X-100 and then rinsing three times in sterile water. Each leaf-half was cut transversely into six 3-mm sections from the basal end upward. The sections from one leaf-half were serially plated on to Schenk and Hildebrandt medium ,,7~containing 30 #M dicamba (SH-30) and denoted as "control", whereas the corresponding sections from the other leaf-half were plated on to SH-30 medium containing ABA concentrations ranging fi~om0.01 to 20.0 #M. Twenty leaf-halves for each ABA treatment level and corresponding controls were cultured in the dark at 22°C. After 1, 3 or 7 days, sections were transferred to SH-30 medium without ABA. Control cultures were also transferred to ti~esh SH-30 medium at the same time the treatment cultures were transt~rred. After 28 days, leaf sections with embryos and/or callus were transferred to SH medium without growth regulators (SH-0) tbr germination. Also at this time, five replications of 10 embryos per treatment having a distinct coleoptile notch were transtErred to SH-0 to determine germination rate. All cultures were maintained in an environmentally controlled incubator on a 16-hr 22°C light-8-hr 15°C dark cycle. Light intensity was 70 # m o l m 2s J. Plantlet formation was recorded

II. Quantification by ELISA Before the initiation of culture on SH-0 medium and at 1, 3, 7 and 14 days thereafter, 20 samples for each culture period were analyzed for endogenous levels of ABA. The basal 3 cm from the two innermost leaves was ground in liquid nitrogen in 10-ml centrifiage tubes, and ABA extraction was performed following the modified scheme ofWEILER, ijSi Alter grinding, 10 ml methanol with 0.1 g 1-l butylhydroxytoluene (BHT) were added per gram of tissue. After a 16-hr extraction at 4°C, samples were centrifuged tbr 5 min at 157 g. The supernatant (containing ABA) was decanted and the volume measured. Methanol content was adjusted to 70°/0 before filtering through a C- 18 Sep-pak. Samples were then dried on a Speedvac concentrator for 4-5 hr. Pellets were resuspended in 1 ml Tris-buffered saline (pH 7.5) and then diluted 1:3 with buffer. Immunoassays for ABA were performed with kits purchased t~om Idetek, Inc., San Bruno, CA. Numbers generated tbr ABA were corrected for losses incurred during extraction. These correction factors were determined by performing extraction on known amounts of ABA standard solutions.

RESULTS

I. Tissue culture A positive effect of ABA on the ability to form embryos was obtained only with Embryogen-P. None of the treatments produced an embryogenic response in 1-39. A 1-day exposure to ABA did not have a significant effect on embryogenesis (Fig. l a). Embryo production was significantly increased with 3-day treatments of 0.01-10.0 #M ABA (Fig. lb); however, 20.0 #M ABA caused a 4.6fold decrease in embryo formation. In 7-day treatments there was a significant decrease in embry-

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endogenous ABA levels and these levels after different periods of culture. T h e initial ABA level was higher in 1-39 than in E m b r y o g e n - P (Fig. 2). However, after 1 day in culture the ABA level had decreased by 12% in E m b r y o g e n - P and by 59% in 1-39. By days 3 and 7, concentrations were 45 and 53°J~ lower in E m b r y o g e n - P and 91 and 93% in 1-39, respectively. After 14 days in culture, there was no significant difference in ABA levels between genotypes.

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Fro. l. Effect of ABA on somatic embryogenesis from leaf segments of Dactylis glomerata. Leaf segments were cultured on Schenk and Hildebrandt medium containing 30 pM dicamba (SH-30) and ABA for the initial 1, 3 or 7 days, (a, b and c, respectively) followed by transfer to SH-30 medium without ABA for the remainder of the 28-day culture period. Control cultures were maintained on SH-30 throughout the experiment. Solid and hatched bars represent control and treatment values, respectively. Asterisk indicates a significant difference between the number of embryos produced on treatment vs control leaf halves at c~ = 0.05 using a one-sided paired t-test. ogenesis with all ABA concentrations except 0.1 /~M (Fig. lc). H. Imrnunoassay I mmunoassay of in vitro cultured leaf tissue indicated significant differences between initial

DISCUSSION

Abscisic acid level appears to be associated with somatic embryogenesis from leaf culture in the embryogenic genotype of orchardgrass. T h e addition of ABA to culture medium (0.01-10.0 #M) for 3 days increased the n u m b e r of embryos formed; however, higher concentrations and/or longer times inhibited embryo formation. These results are in agreement with those of RAJASEKARAN et al. 16i who reported that timing and duration of exogenous ABA application also had a marked effect on embryo formation and growth in napiergrass (Pennisetum purpureum Schum.). The lack of an embryogenic response in the nonembryogenic genotype m a y be due to the rapid loss of ABA during culture rather than initial concentration. RAJASEKARAN et al. (5~ reported that higher levels of ABA in basal leaf sections ofnapiergrass were correlated with a greater embryogenic response. Addition offluridone, an ABA synthesis inhibitor reduced embryogenesis in that system. (6' In the present experiments, the addition of ABA to the medium was not effective in inducing embryogenesis in the nonembryogenic genotype. Possible explanations are: (i) ABA was not taken-up effectively by the leaf tissue; (ii) it was not transloeated to effective sites; or (iii) there are additional fhctors (hormonal or otherwise) needed to elicit the response. Additional experiments with orchardgrass involving quantification of endogenous levels in leaf tissue after culture for various periods on medium containing ABA might help define the role of this hormone in somatic embryogenesis. However, the maintenance of high ABA levels during culture of E m b r y o g e n - P on medium not containing ABA supports the hypothesis that somatic embryogenesis m a y be a response to senescence.

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FIG. 2. Level of ABA (pmol g L ii'esh weight of tissue) in cultured leaf segments of Dactylis glomerata as shown by immunoassay. Leaf segments from embryogenic and nonembryogenic genotypes were sampled at culture initiation and at days 1, 3, 7 and 14 thereafter. Small squares above and below data points indicate standard error values.

REFERENCES

1. CONGERB. V. and HANNINGG. E. (1991) Registration of Embryogen-P orchardgrass germplasm with high capacity for somatic embryogenesis from in vitro cultures. Crop Sci. 31,855. 2. HANmNG G. E. and CONGER B. V. (1982) Embryoid and plantlet formation from leaf segments of Dactylis glornerata L. Theor. appl. Genet. 63, 155-159. 3. HANNINGG. E. and CONGERB. V. (1986) Factors influencing somatic embryogenesis from cultured leaf segments of Dactylis glomerata. J. Plant Physiol. 123, 23-29. 4. KELLYM. O. and DAVIESP.J. (1988) The control of whole plant senescence. CRC Crit. Rev. Plant Sci. 7, 139-173. 5. RAJASEKARAN K., H E I N M . B., DAVIS G . C . , CARNeS M. G. and VamL I. K. (1987) Endogenous growth regulators in leaves and tissue cultures of Pennisetum purpureum Schum. J. Plant Physiol. 130, 13 25. 6. RAJASEKARAN K., HEIN M. B. and VASIL I. K. "(1987) Endogenous abscisic acid and indole-3acetic acid and somatic embryogenesis in cultured leafexplants ofPennisetum purpureum Schum. Effects in vivo and in vitro of glyphosate, fluridone, and paclobutrazol. Plant Physiol. 84, 47-51.

7. SCHENK R. U. and HILDEBRANDTA. C. (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot. 50, 199-204. 8. SONOSTADD. D., PETRACEKP. D., SAMSC. E. and CONGER B. V. (1989) Effect of 1-aminocyclopropane-l-carboxylic acid and aminoethoxyvinylglycine on ethylene emanation and somatic embryogenesis from orchardgrass leaf cultures. Plant Cell Rep. 7, 677-679. 9. THIMANNK. V. (1980) The senescence of leaves. Pages 85-115 in K. V. THIAMANN,ed. Senescencein plants. CRC Press, Boca Raton, FL. 10. TRIGIANOR. N. and CONGERB. V. (1987) Regulation of growth and somatic embryogenesis by proline and serine in suspension cultures of Dactylis glomerata. J. Plant Physiol. 130, 49-55. 11. TRIGIANOR. N., CONGERB. V. and SONGSTADD. D. (1987) Effects ofmefluidide and dicamba on in vitro growth and embryogenesis of Dactylis glomerata (orchardgrass). J. Plant Growth Regul. 6, 133 146. 12. TRIOIANO R. N., MAY R. A. and CONGER B. V. (1992) Reduced nitrogen influences somatic embryo quality and plant regeneration from suspension cultures oforchardgrass. In Vitro Cell. dev. Biol. 28P, 187 191. 13. VASIL I. K. (1987) Developing cell and tissue

ABA AND EMBRYOGENESIS IN DACTYLIS culture systems for the improvement of cereal and grass crops. J. Plant Physiol. 128, 193-218. 14. VASIL I. K. (1988) Progress in the regeneration and genetic manipulation of cereal crops. Bio/ Technology 6, 397402. 15. WEmER E. W. (1984) Immunoassay of plant growth regulators. A. Rev. Plant Physiol. 35, 8595.

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16. WENCKA. R., CONGER B. V., TRIGIANOR. N. and SAMS C. E. (1988) Inhibition of somatic embryogenesis in orchardgrass by endogenous cytokinins. Plant Physiol. 88, 990-992. 17. WILLIAMS E. G. and MArlESWARAN G. (1986) Somatic embryogenesis: factors influencing coordinated behaviour of cells as an embryogenic group. Ann. Bot. 57, 443-462.