IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY Volume 21, Number 10, October 1985 �9 1985 Tissue Culture Association, Inc.

S C R E E N I N G F O R D R O U G H T T O L E R A N C E IN S O R G H U M U S I N G C E L L C U L T U R E

R. H. SMITH, S. BHASKARAN, AND F. R. MILLER

Department of Soil & Crop Sciences, Texas A&M University, College Station, Texas 77843

(Received 3 September 1985; accepted 3 September 1985)

SUMMARY

Callus growth from 10 cultivars of Sorghum bicolor ILA Moench was measured with increasing levels of polyethylene glycol (PEG} as an osmoticum in the medium t o determine whether differences among these cultivars at the cellular level in response to osmotic stress existed. These cellular ratings were compared to field ratings from the i0 tolerant-to-susceptible cultivars when grown under drought conditions to determine whether cellular ratings corresponded to differences in drought tolerance at the plant level. Callus cultures were grown on Murashige and Skoog inorganic salt formulation plus vitamins, 2,4-dichlor0phenoxyacetic acid (2,4-DL kinetin and sucrose, supplemented w i t h 0 to 25% lwt/vol) PEG corresponding to --0.2 to --1.62 n P a osmotic potential. Results suggest that PEG-induced osmotic stress on callus cultures can be used to screen sorghum cultivars for potential early field ~prefloweriug} drought tolerance. This implies that at least a component of the early field drought tolerance in sorghum may have a cellular basis.

Key words: callus of sorghum; germplasm screening; osmotic stress; tissue culture; Sorghum bicolor (L) Moench.

INTRODUCTION

Plants exposed to drought stress respond by various resistance mechanisms, and in any species the relative importance of different components can change. Plants can minimize water stress by avoidance, postponement, and tolerance (10). These mechanisms range from whole-plant characteristics such as life-cycle timing Imaturity), deep root systems, leaf orientation, cuticle thickness and stomatal closure, to cellular-level functions (osmoregulation}. A single trait alone may not ensure successful survival; however, improvement in any one aspect may enhance overall water stress resistance. It seems likely that the cellular-level components of drought-tolerance mechanisms are important, and improve- ment at this level could have a positive impact on whole plant tolerance.

Tissue culture offers opportunities to study cellular- level responses to osmotic stress and possibly identify cell lines that differ in osmotic adjustment capabilities. Cellular-level tolerance might also be most amenable to genetic manipulation because only a few genes for metabolic processes involved in osmoregulation may be involved ~9D. Additional benefits would include develop- ment of methods to evaluate and screen potentially tolerant germplasm for drought tolerance, assuming that a similarity exists between cellular-level responses and whole-plant responses under field conditions. The ability to regenerate plants from tolerant cell lines and obtain

541

enhanced tolerance at the plant level would be an additional advantage. Potential physiological studies Of enzyme act ivi ty , osmoregulatory compounds, nitrogen metabolism, and genetic markers with a tissue culture system could also advance the fundamental understand- ing of water stress.

Several studies support correlations between whole- plant and cell-culture responses for salt tolerance. Barlass and Skene I1) found relative tolerance of graPe (Vitis species) cultivars to salt to be the same in vitro and for whole plants. Likewise, Orton (13~ determined for cultivated barley (Hordeum vulgare L.~ and a wild relative that salt tolerance at the cellular level is similar to that at the whole-plant level. Nabors et al. (12) selected salt-tolerant tobacco cell lines in culture, and plants regenerated transmitted tolerance to subsequent genera- tions.

Polyethylene glycol is assumed to be a nonpenetrating osmotic agent that lowers the water potential of the medium and has been used to simulate drought stress in plants (4). This assumption has been questioned because Yaniv and Werker (17) have demonstrated that PEG- induced water stress in Solanaceae species resulted in PEG secretion from the leaves. Bressan et al. (4,5) and Handa et al. 17,8j have reported using PEG to select tolerant tomato (Lycopersicon esculentum Millj cell lines and indicated that PEG does not contribute to the osmotic adjustment of selected cells (7). The tolerant cells grew better than cells never exposed to PEG, but lost resistance

542 SMITH ET AL.

in a medium lacking PEG (4). These studies support the use of PEG to induce water stress at the cellular level.

This study investigated the use of PEG-induced water stress on 10 cultivars of Sorghum bicolor (L.} Moench at the cellular level to establish relative ratings from susceptible to tolerant for osmotic stress. Results were compared to field evaluation of these cultivars to drought stress. Whole-plant drought tolerance undoubtedly is a very complicated interaction of genotypes, environmental factors, and varied mechanisms at the plant level; however, if a significant comparison can be found between whole-plant response to drought stress and a cellular-level repsonse, perhaps a useful in vitro method to screen sorghum germplasm for osmotic tolerance could be available for applied breeding programs. Additionally, this system could have tremendous utility in physiological and biochemical studies of this compo- nent in drought tolerance.

MATERIALS AND METHODS

Seed-derived callus was established from Sorghum bicolor (L.} Moench , cultivars RTx430, 1790E, B-35, RTx7078, RTx7000, RTx432, BTx3197, BTx623, BTx378, and R9188, in liquid medium containing the Murashige and Skoog inorganic salts (11), 20 g L -~ sucrose, and in milligrams L -t: 0.1 th iamine 'HC1, 2 glycine, 0.5 nicotinic acid, 0.5 pyridoxineeHC1, 100 i-inositol, 2.5 2,4-D, and 0.05 kinetin (2,16). Seeds were surface- disinfected in 20% (vol/vol) Clorox for 15 rain and rinsed three times in sterile water. Cultures were incubated at 27 ~ C, with photon flux density of 24 gmol m-2s -1 and a ]6-h photoperiod. Callus obtained was subcuhured on the same medium with PEG (molecular weight 8000) treatments at 0, 5, 10, 15, 20, and 25% (wt/vol) cor responding to - -0 .2 , - -0 .35 , - -0 .5 , - -0 .75 ,

1.15, and --1.62 MPa osmotic stress. All callus was initiated at the same time; therefore, the physiologi- cal age of the cultures was identical. All cultures were grown on a filter paper support in a liquid medium. Ten separate replications of each treatment were individually weighed on a weekly basis (0 to 8 wk). Weights were asep- tically measured rapidly under a laminar air flow hood on a

top-loading Mettler balance (accurate to + 0.1 mg) attached to a programmed computer which printed out weights. In every experiment the starting callus weight was 250 -1- 5 rag, and each growth curve represented an average of individual weights. The experimental procedure was a 2-factor factorial in a completely randomized design. T h e data were analyzed accordingly, except that to reduce variability among cultivars, growth of the tissue on the control medium was used as a covariate for each cultivar (6). The cuitivar by treatment interactions were highly significant (P <0.0001} in all cases. The Scott-Knott multiple comparisons procedure (6} was used to separate cultivars within treatments.

Field ratings for both early and late drought tolerance are shown in Table 1. These ratings were established over a 10-yr period through the sorghum breeding programs directed by Drs. F. Miller and D. Rosenow at Texas A&M University field locations at Lubbock, Big Springs, Temple, Chillicothe, Amarillo, and College Station. Visual ratings of 1 to 5 (1 = good tolerance and 5 = death} were used to establish the relative ratings for these cuhivars (14,15). Visual observations for preflowering stress symptoms included leaf rolling, excessive leaf erectness, leaf bleaching, leaf tip and margin burn, delayed flowering, poor head exsertion, head and floret abortion and blasting, and reduced head size. Posfflower- ing stress symptoms included premature plant (leaf and stem) death or senescence, often accompanied by charcoal rotted stalks and stalk lodging and sometimes seed size reduction.

RESULTS

Field ratings identified RTx430, RTx7078, RTx7000, and BTx623 to be drought tolerant during germination and until preanthesis growth (Table 1). However, their tolerance (except RTx430} to drought during grain fill and maturation is poor. Cultivars B-35 and 1790E are the most drought tolerant during late-field drought condi- tions (posfflowering}.

Callus growth curves of three cultivars representing good growth (RTx430), intermediate (RTx432), and poor (BTx3197) growth are presented in Fig. 1. Within each

TABLE 1

FIELD RATINGS OF EARLY AND LATE-DROUGHT TOLERANCE FOR 10 SORGHUM CULTIVARS AT LUBBOCK, BIG SPRING, AMARILLO, CHILLICOTHE, AND COLLEGE STATION, TX. OBSERVATIONS

MADE BY DRS. D. ROSENOW, L. CLARK, AND F. MILLER.

Excellent Very Good Good Fair Very Poor Susceptible

Field BTx623 RTx432 1790E B-35 BTx378 Early RTxT000 BTx3197 Drought RTx7078 R9188 (Pre-fiowering) RTx430

Field B-35 1790E RTx430 RTx7078 BTx623 BTx3197 Late RTx432 RTx7000 BTx378 Drought R9188 (Post-flowering)

SCREENING FOR DRAFT TOLERANCE IN VITRO 543

indiv idual euh iva r s tat is t ical analyses es tabl ished differ- ences in growth in response to increasing P E G to be s ignif icant a t P <0.0001.

Stat is t ical evaluat ion of the da ta a t P = 0.01 and 0.05 were very similar , and T a b l e 2 shows, as indicated by a slash ( /) , the s ignif icant groupings of the cuh ivars at P = 0.05. In this table the cuh ivars are represented by a n u m b e r {see Tab le 2 for key). C u h i v a r RTx430 = 1 grew

significantly be t te r in compar i son to other cul t ivars a t all t r ea tmen t s except af ter 4 wk on 15, 20, and 25% P E G . C u h i v a r s RTx7000 = 5, and RTx7078 = 4 general ly were the next bes t in growth unde r osmotic stress. T h e cul t ivars t h a t showed the poorest response unde r osmotic stress were BTx3197 = 7, R9188 = 0, and B-35 = 3. At 20% P E G af ter 5 wk in culture, RTx430 = 1, RTx7078 = 4, RTx7000 = 5, and BTx623 = 8 grew signif icantly

2.21. - .

2.01 R T x 4 3 0 on PEG

1.8

1 .6

A r 1.4 E 0 3

1.2 .C: t3~

~ 1 , o

U -

0.6

0.4

0.2

06I_ RTx432 on PEG

0.51

0.4

O~ v

~ 0.3

oJ

0.2

~, 1 0.1

0 1 2 3 4 5

Time ( w e e k s )

6 7 8 0 1 2 3 4 5 6 7 8 Time (weeks )

~ 0 . 7

~ 0.6

~ 0 . 5

~ 0 . 3

0 . 2

0 .1

B T x 3 1 9 7 on PEG

i i t i i

1 2 3 4 5 6 7 8

T i m e ( w e e k s )

FIG. 1. A-C, callus fresh-weight growth curves for three sorghum cultivars over an 8-wk period on increasing levels of PEG. O = control, A = 5% PEG, �9 = 10% PEG, )< = 15% PEG, [] = 20% PEG, �9 = 25% PEG.

544 SMITH ET AL.

TABLE 2

STATISTICAL EVALUATION USING THE SCOTT-KNOTT PROCEDURE AT P = 0.05 OF THE 10 SORGHUM CULTIVARS AT INCREASING LEVELS OF PEG OVER AN 8-WK GROWTH PERIOD IN VITRO ~

PEG, %

Week 0 5 10 15 20 25

1 1/52/7493/086 1/4583/92/067 15/4/938/2670 1/542/8396/07 1/45/3829/760 1/85293/4607 2 1/5/27/409836 1/4859/32067 1/54/98632/70 1/452/839/670 1/45/8293/760 1/82593/3607 3 1/5/72/4098/63 1/4598/02637 1/54/98/62307 1/542/869307 1/45/8293/670 1/852943067 4 1/57024/986/3 1/458/906/237 1/54/986/2037 1542/869307 1/458/239670 1852/493076 5 1/57486209/3 1/458/690/237 1/54/9862037 1543/896307 1458/293670 1852493067 6 1/5749/82063 1/4/85/960/327 1/54/9862037 15248/96307 1458923/670 1582943067 7 1/75042/6983 1/4/58/609/327 1/54/9086237 152/4869370 1458329670 1528943067 8 1/072456/893 1/4/58609/327 1/549/86237 152/8469370 1 5 4 8 3 9 2 7 6 152894367

(NoO) {NoO~ ~No0~

~The numbers separated by / indicate significant grouping differences. The numerical codes for cultivar iden- tification are: 1 ---- RTx430, 2 = 1790E, 3 = B35, 4 = RTx7078, 5 ---- RTx7000, 6 ---- RTx432, 7 = BTx3197, 8 ---- BTx623, 9 = BTx378, 0 = R9188.

better than the other cultivars. It is interesting that these four eultivars are also the ones that are rated as very good under early field drought conditions.

DISCUSSION

It is extremely difficult to make direct comparisons between whole-plant drought tolerance and osmotic tolerance at the cellular level. Whole-plant drought tolerance is a muhigenic trait involving root structure, leaf anatomy, environmental interactions on genotype, etc., as well as mechanisms at the cellular level potentially involving osmoregulatory compounds, compartmenta- tion, and most likely other metabolic events. Because of the diverse germplasm in sorghum it seems reasonable to expect that for some cultivars the cellular-level compo- nents may vary from being significant contributors to overall whole-plant tolerance to perhaps playing a very minor role.

In general, among these 10 cultivars tested at the cellular level for differences in response to osmotic stress, it seems to be more than coincidental that of the top four under field conditions of early drought tolerance (BTx623, RTx7000, RTx7078 and RTx430J, three (RTxT000, RTxT078, and RTx4301 are also the best at the cellular level for osmotic tolerance. The cultivars at the other extreme (BTx378, BTx3197, R9188, and B-35~ are very poor to susceptible under field conditions of early drought. At the cellular level two of these, R9188 and BTx3197, consistently showed the poorest growth response. For the cultivars that perform best under late field conditions of drought, there seemed to be no association with the cellular-level ratings. This may suggest that an important component of the ability for field-grown sorghum to cope with early drought tolerance is a cellular-level mechanism. This also may imply that cellular-level drought tolerance mechanisms for late tolerance may not be as significant as other components of drought tolerance. Although salt-tolerance mecha- nisms probably differ in many respects from drought

tolerance, others (1,13) have also established a similarity between field and cellular-level tolerance to salt.

The strong similarity between cellular-level osmotic tolerance and early field drought tolerance established in these studies suggests that the technique can be useful to rapidly evaluate and screen sorghum germplasm. A rapid screen would involve induction of callus from RTx430, RTx7000 and RTx7078 (to be used as controlM and the cultivars to be screened, which can take 6 to 8 wk. Subculture of this callus onto a media containing 0, 15, and 20% PEG (which represent significant osmotic stress levels~ plus collection of fresh weights of these cultures at 0, 10, 20 and 30 d should establish whether the cultivars significantly grow as well as the control group or not. Separation of the cultivars on this basis should have a very high probability of establishing those which should perform well under early field drought stress. Cellular- level screening on PEG could also be used to select for tolerant cells and subsequent regeneration of plants that might express enhanced tolerance to drought stress. The 10 cuhivars used in this study so far will not regenerate plants from callus cultures.

Further studies of the physiological and biochemical processes associated with cellular-level tolerance to PEG are in progress (3D. These studies may provide valuable information on osmoregulation at the cellular level and further insight into understanding drought mechanisms under field conditions.

REFERENCES

1. Barlass, M.; Skene, K. G. M. Relative NaC1 tolerances of grapevine cultivars and hybrids in vitro. Z. Pflanzenphysiol. Bd. 102:147-156; 1981.

2. Bhaskaran, S.; Smith, R. H.; Schertz, K. Sodium chloride tolerant callus of Sorghum bicolor (L.) Moench. Z. Pflan- zenphysiol. Bd. 112:459-463; 1983.

3. Bhaskaran, S.; Smith, R. H.; Newton, R. J. Physiological changes in cultured sorghum cells in response to induced water stress I. Free proline. Physiol. Plant 79:266-269; 1985.

SCREENING FOR DRAFT TOLERANCE IN VITRO 545

4. Bressan, R. A.; Hasegawa, P. M.; Handa, A. K. Resistance of cultured higher plant cells to polyethylene glycol-induced water stress. Plant Sci. Lett. 21:23-30; 1981.

5. Bressan, R. A.; Handa, A. K.; Handa, S.; et al. Growth and water relations of cultured tomato cells after adjustment to low external water potentials. Plant Physiol. 70:1303-1309; 1982.

6. Gates, C. E.; Bilbro, J. D. Illustration of a cluster analysis method for mean separation. Agro. J. 70:462-465; 1978.

7. Handa, A. K.; Bressan, R. A.; Handa, S.; et al. Characteristics of cultured tomato cells after prolonged exposure to medium containing polyethylene glycol. Plant Physiol. 69:514-521; 1982.

8. Handa, S.; Bressan, R. A.; Handa, A. K.; et al. Solutes con- tributing to osmotic adjustment in cultured plant cells adapted to water stress. Plant Physiol.. 73:834-843; 1983.

9. Heikkila, J. J.; Papp, J. E. T.; Schultz, G. A.; et al. Induction of heat shock protein messenger RNA in maize mesocotyls by water stress, abscisic acid, and wounding. Plant Physiol. 76:270-274; 1984.

10. Kramer, P. J. Water stress research--progress and problems. In: Randall, D. D., ed. Current topics in plant biochemistry and physiology. Columbia, MO: Univ. of Missouri Press; 1983:129.

11. Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15:473-497; 1962.

12. Nabors, M. W.; Gibbs, S. E.; Bernstein, C. S.; et al. NaC1- tolerant tobacco plants from cultured cells. Z. Pflanzenphysiol. Bd. 97:13-17; 1980.

13. Orton, T. J. Comparisons of salt tolerance between Hordeum vulgare and H. jubatum in whole plants and callus cultures. Z. Pflanzenphysiol. 98:106-118; 1980.

14. Rosenow, D. T.; Clark, L. E. Drought tolerance in sorghum. Am. Seed Trade Assoc. Proc. 36:18-30; 1981.

15. Rosenow, D. T.; Quisenberry, J. E.; Wendt, C. W.; et al. Drought-tolerant sorghum and cotton germplasm. Agric. Water Manage. 7:207-222; 1983.

16. Smith, R. H.; Bhaskaran, S.; Schertz, K. Sorghum plant regeneration from aluminum selection media. Plant Cell Reps. 2:129-132; 1983.

17. Yaniv, Z.; Werker, E. Absorption and secretion of polyethylene glycol by solanacous plants. J. Exp. Bot. 34{148):1557-1584; 1983.

The authors thank Dr. C. Gates and Mr. Ron Berdine, Statistics Institute, Texas A&M University, for their assistance in statistical analysis of the data, This study was supported by U.S. Agency |or International Development Grant AID/DSAN/XII /G-0149, and USDA Competitive Grants Program.