Plant Cell Reports

Plant Cell Reports (1989) 8:411 414

© Springer-Verlag 1989

Development of salt tolerant lines of KDML and LPT rice cultivars through tissue culture M. Vajrabhaya, T. Thanapaisal, and T. Vajrabhaya Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Received April 5, 1989/Revised version received July 8, 1989 - Communicated by A. R. Gould

ABSTRACT

MATERIALS AND METHODS

Salt tolerant lines of indica rice (Oryza sativa L.) were selected out of KDML and LPT cultivars. The first selection was made in vitro by incorporating i or 2% NaCI in the culture media. Embryogenic cal!i from mature embryo were subjected to a salt stress for four weeks. Regeneration rates after salt stress were reduced to 0.076% or less as against regeneration rates of 8.3 to 30% normally obtained for non-stressed conditions. Seedlings of regenerants and of f o l l o w i n g generations were treated with 0.5% NaCI in water culture for four weeks. Definite salt tolerance of the progenies of s e l e c t e d and u n s e l e c t e d plants appeared in both cultivars. The best survival rate of line LPT 171 in R 3 was 94.3% w h i l e only 2% of the control survived. The result of the fourth generation was similar to the third.

Callus induction and plant rei~eneration

ABBREVIATIONS

KDML LPT MS WP RI R2

= = = = = = =

Cultivar Kao-Dawk-Mali; Cultivar Leung-Pra-Tiew; Culture medium (Murashige and Skoog, Nutrient solution (Bentley, 1959); Regenerated plants from callus; Progenies of ~ ; Progenies of R I. . .etc.

1962);

INTRODUCTION

Biotechnological approaches in d e v e l o p i n g salt tolerant cereal crops have been sought for several years. The main problem encountered has been the low rate of plant regeneration from cell aggregates d e r i v e d from e x p l a n t s w h i c h can rarely supply materials for mass selection. Moreover, the mechanisms controlling salt tolerance in rice are not clearly understood (Greenway and Munns, 1980; Tal, 1984; Yeo and Flowers, 1984). This paper covers some of the procedures used in the selection in vitro and in natural conditions, both of which are e s s e n t i a l for obtaining salt tolerant lines. The results of a selection going up to the fourth generation from regenerated plants are reported in this study.

This research was sponsored by the United States A g e n c y for I n t e r n a t i o n a l Development, Washington D.C., Grant no. 936-5542-C~-SS-3037-00

Offprint requests to: M. Vajrabhaya

Callus induction was done by culturing over 450,000 dehusked seeds on MS medium with i mg/l 2,4-D and 0.3 mg/l kinetin in the dark for two to four weeks. 0nly large and h e a l t h y e m b r y o g e n i c calli w e r e severed and cultured on a modified White's medium with 200 mg/l (NH4)2S04, 100ml/l coconut water, 3 mg/l kinetin, I mg/l IAA for plant regeneration. The culture vessels were exposed to light 16 hrs. a day at 24 + 3 °C. Salt selection in vitro Selection was done in the callus stage two to four weeks after t h e first c u l t u r e in the callus induction medium. Media and physical environments u s e d are m e n t i o n e d above according to stages of development of the tissues except for the presence of i or 2% NaCl. The length of treatment was four weeks for all stages of growth. Salt selection in natural conditions The water culture technique used in the selection was similar to that of Yoshida et al. (1983) with the difference that the plants in this selection w e r e grown in n a t u r a l c o n d i t i o n s u n d e r a rain shelter with 50% light transmission instead of a growth cabinet. One t h r e e - l e a f stage s e e d l i n g of s e l e c t e d and unselected regenerants ( ~ ) or their progenies was i n s e r t e d in holes m a d e in i cm-thick styrofoam plates and held in place by a piece of synthetic sponge wrapped around the stem. This twenty fivehole styrofoam raft was then floated in a round three-liter plastic tray filled to a height of 10 cm w i t h a m o d i f i e d WP n u t r i e n t s o l u t i o n (Bentley, 1959). When the seedlings reached a four to fiveleaf stage, the nutrient solution was replaced by a n o t h e r freshly prepared solution with 0.5% NaCI added. In the meantime, the level of the solution was k e p t constant by either adding tap water or adding a salinized nutrient solution according to the electrical conductivity of the solution which was maintained to 9 - 10 mmho/cm at 25 °C. Dead plants were removed every week and not more than 2% of the total population were selected for growing to maturity at the end of the fourth week.

412 Each selected plant grew to maturity in a 28 cm diameter jar filled with soil with at least a I cm of water above the soil surface.

onto the regeneration medium. Yamada et al. (1983) also reported that four subcultures in media with different concentrations of seawater were more than enough to produce salt-tolerant cells.

RESULTS

Selection in vitro After f o u r w e e k s of salt s e l e c t i o n at 1 and occasionally 2% NaCI level, 38 of the calli (in a population over 50,000) of each cultivar survived. Upon transferring these calli to the regeneration medium, most turned brown and died within two weeks, the rest continued to grow and finally developed into shoots or plantlets. The average rate of regeneration after salt stress at this stage was found to be 0.076% as against 8.3 for KDML and up to 30% for LPT in the salt free medium. Many shoots and even plantlets were lost during the last stages of growth in vitro and again in the early stages of water culture. A total of three regenerated plants of KDML and nine of the LPT were obtained from in vitro selection w i t h salt and w e r e grown to maturity. Drastic changes in morphology were not observed in these plants. Selection in natural conditions After one week of salt stress (0.5% NaCl) in a water culture, the tips of lower blades began to dry up. Severe symptoms developed as the drying of leaves moved up in the second week; at the end of the third week, more than half of the seedlings from both breeder seeds and ~ died (characterized by loss of green color in the leafblades and leaf sheath). At the end of the fourth week, only 4 to 4.5% of the KDML seedlings from breeder seeds survived while many seedlings of the R 0 both from in vitro showed definite salt tolerant traits with green leaves and near normal height with a survival rate of 19.8% (Table i, Fig. 1 and other data not shown here).

It is assumed that a small group of salt tolerant cells results from nuclear gene mutation with and w i t h o u t s e l e c t i o n pressure, and forms sectorial chimera in the early stages of callus induction. The rate of mutation is estimated to be 10 -7 - 10 -8 in the callus culture (Maliga, 1980), and the rate of double mutation occurring in the same cell is e s t i m a t e d to be e x t r e m e l y low (10 - 1 4 10-16) o However, Sun et al. (1981) found a n u m b e r of s o m a c l o n e s w i t h two or more traits that varied simultaneously within a single plant. One should be aware of multiple mutations that add undesirable characters to the salt tolerant regenerants (R0). The salt tolerant lines (R1 and after) obtained by this method should be evaluated carefully for yield, disease resistance and other characteristics that might arise during tissue culture. A chance of adding beneficial characteristics to salt tolerant lines should not be overlooked. Wide variations in salt tolerance found in the first generation seedlings (R I) of KDML arising from unselected cultures indicate that the variation in salt tolerance characters occurs spontaneously (data not shown here). The most tolerant line gave a 19.8% survival rate at the end of the fourth week, and other lines with survival rates above 15% were grown and produced R 2 and R 3 (Table I). However, all of the R 1 derived from salt selected ~ of both cultivars are salt tolerant as expected, but the degrees of tolerance varied appreciably (unpublished data). The selected line no. 69 of KDML showed similar degrees of salt tolerance to the unselected lines nos. 161 and 176 in R 2 and R 3 (Table I).

DISCUSSION

The R 3 and R 4 plants from the most tolerant R 2 of L P T no. 171 s h o w e d progressive t o l e r a n c e by selection (Table 3). Most of the plants were affected b y NaCI similarly as those seedlings from the breeder seeds or from other tolerant and semitolerant lines but to lesser degrees during the first two weeks of treatment (Fig° I A). It is interesting to note that this particular line (no. 171) exhibits the ability to recover while only some members of line no. 144 have the ability to recover slowly towards the end of the fourth week (Fig. i B). At this period, the survival rate of no. 171 line was 94.3% with their appearance being similar to the control in color of leaves, size of stems and height while only 2 to 3% of the seedlings of the breeder seeds barely survived with small plants and yellowish green leaves (Fig, i B, Table 3). Other seedlings of the selected lines were intermediate b o t h in the rate of survival and the ability to recover.

The aim of this work was to introduce a heritable salt tolerant character into the established rice cultivars. Therefore, a combination of short term callus culture method and one step high level stress were adopted to reduce the chance of multiple gene mutations occurring due to prolonged culture both before or during salt stress. Our salt selection was done by cultivating the selected embryogenic calli in a medium containing 1 or 2% NaCl for four weeks only, whereas Yoshida et el. (1983) divided the calli into small pieces and selected them in 1.5% NaC1 for four weeks. This treatment was repeated three to six times before transferring them

There have been several reports on salt-tolerant cell-llnes in higher plants but most disappeared in t h e w h o l e plant r e g e n e r a t e d from them. Some regenerants of alfalfa showed many lethal genetic changes w h i c h could not be e v a l u a t e d for salt tolerance (Stavarek and Rains 1984), and salt tolerant lines of flax regenerated from callus were found to fall in recent i n t e n s i v e field tests (Rowland et al., 1989). However, Nabors et al. (1980) reported that F 2 generation of tobacco plants obtained from salt tolerant cell lines still inherited salt tolerant characters. Here, we report the progressive salt tolerance of rice to the third

The R 2 seedlings from selected R 1 seedlings showed i n c r e a s e d salt tolerance, giving 19.8 to 53.3% survival rates in KDML and 29.9 to 63.9% in LPT (Table 1 and 2). The survival of the R 3 was very much the same as each of the R 2 parents. On the contrary, the R 3 seedlings of the most tolerant parent (no. 171) of LPT gave 94.3% survival rate in a p o p u l a t i o n of 1031 seedlings (Table 3). Salt tolerance of R 4 from these parents still produced 92.5% survivors in a population of 200 which was the only line g i v i n g survival rate over 90% in two successive generations (Table 3). Deviation of salt-selected lines in terms of general characters, seed set, percentage germination, and of vigor of s e e d l i n g s from the original cultivars were not observed.

413 generation which generation.

still persists

through the fourth

The marked increase in survival of the R 3 over R 2 generations of the tolerant lines reflects the occurrence of either a polygenic or a multiallelic nature of salt tolerant genotypes inherited through sexual reproduction (Moeljopawiro and Ikehashi, 1981). If this is the case, several generations would need to be grown under continuous selection pressures to establish suitable recombinations. However, the R 3 of KDML in which the original cultivar is more tolerant than the LPT, did not show any i n c r e a s e in salt t o l e r a n c e over the R 2 generation. There might simply be fewer genes involved for salt tolerance than those in the LPT or there may be lack of other complementary genes which intensify the action of existing genes to increase salt tolerance or salt avoidance.

We did not study the details of salt tolerance and whether it occurs in the cell, the organ, or at the plant l e v e l or w h e t h e r the young plant that tolerates NaCI stress will survive in the field through maturity as a result of several other salts being present in the soils. However, salt tolerance in rice is likely to be at the cell level because progenies of the selected regenerants arising from callus are salt tolerant. This work is in agreement w i t h W o n g et al. (1986) who worked on rice. However, other plants require a certain level of cell organization to deal with salts. Stavarek and Rains (1984) found that rice cells selected for their tolerance to NaCI were also tolerant to all other salts; this might hold true at the plant level. Our finding that increase in salt tolerance arises in vitro and can be intensified through selection have shed more light into the possibility of developing a new rice cultivar with only single desirable character added to an established cultivar through tissue culture. Table 1

Line No.

Control 1 Control 2

Survival of salt tolerant lines of KDML in water culture which 0.5% NaCI added.

bThe electrical mmho/cm at 25 ° C.

Table 2

99.5 99.0

90.5 89.5

41.0 39.5

4.5 4.0

1695

92.7

63.5

38.8

19.8

14 (R2) 14 (R3)

325 200

i00.0 99.0

88.3 87.5

61.8 60.0

22.1 25.5

26 (R2) 26 (R3)

139 200

I00.0 i00.0

72.7 75.5

65.5 68.0

29.5 30.5

63 (R2) 63 (R3)

25.7 200.0

97.7 99.5

89.9 87.5

69.3 70.5

38.I 37.5

176 (R2) 176 (R3)

93 200

98.9 99.5

93.5 90.5

81.7 79.5

49.5 50.0

161 (R2) 161 (R3)

102 200

98.0 98.0

89.2 89.5

66.7 65.5

50.0 51.5

69 (R2) 69 (R3)

291 200

93.1 93.0

69.1 69.0

53.3 54.5

99.0 97.0

conductivity

ranged

from

9-10

Survival rate of R 2 generation of LPT in water culture with 0.5% NaCI added°

Number Line No, of Plants

ist wk.

Survival rate (%) 2nd wk. 3rd wk. 4th wk.

Control

200

92.5

77.5

27.0

3.0

165

498

I00.0

80.1

55.8

29.9

127

339

97.3

87.9

77.0

31o6

134

97

I00.0

98.9

53.6

32.9

152

537

99.8

89.0

50.8

33.9

129

532

99.2

93.6

78.2

38.2

144

327

98.2

94.8

64.5

52.3

117

734

98.2

94.8

81.9

56.3

171

537

i00.0

87.0

80.3

63.9

aAll salt tolerant lines originated from Ro plants regenerated from calli cultured in medium with 1% NaCI added except line no. 171 which was a callus treated with 2% NaCI. bThe electrical conductivity mmho/cm at 25 ° C.

Table 3

Number Survival rate (%) of Plants ist wk. 2nd wk. 3rd wk. 4th wk.

200 200

1 (RI)

asalt tolerant lines were selected from stressed and non-stressed calli; No. 69 was the only line from stressed callus.

ranged

from

9-10

Survival of R 2, R 3, and R 4 generations of line no. 171 of LPT in water culture with 0.5% NaCl added.

Number Line No. of Plants

Ist wk.

Control 1 R2

200 537

92.5 i00.0

77°5 87.0

27.0 80.3

3.0 63.9

Control 2 R3

200 1031

93.0 99.2

73.0 98.1

23.0 95.9

2.0 94.3

Control 3 R4

~00 200

92.5 99.0

75.0 97.0

23.5 96°0

2.0 92.5

aThe electrical mmho/cm at 25 ° C.

Survival rate (%) 2nd wk. 3rd wk. 4th wk.

conductivity

ranged

from

9-10

414 Greenway H, Munns R (1980) Ann Rev Plan Physiol 31: 149-190 Maliga P (1980) In: Vasil I K (ed), Int Rev Cytol Suppl, Vol IIA, Academic Press, New York London Toronto Sydney Sen Francisco, pp 225250 Moeljopawire S, Ikehashi H (1981) Euphytica 30: 291-300 Murashige T, 473-497

Skoog F

(1962) Physiol Plant

15:

Nabors MW, Gibbs SE, Bernstein Cs, Meis ME (1980) Z Pflanzenphysiol 97: 13-17. Rowland GG, McHughen A, McOnie C (1989) Can J Sci 69: 49-60

LPTBR~EZRSEED

LPT,FEEE~ZS[D

~WKSCONTEOL

~WKS~ 0 ~

LFT~R~I.R ~WKSi~&~N~

LPT~C171Na2~

Stavarek SJ, Rains DW (19B4) In: Staples RC, Toenniessen GH (ads), Salinity Tolerance in Plants, John Wiley and Sons, New York Chichester Brisbane Toronto Singapore, pp 321-334

ff~KSi~0 ~

FIGURE i. R 4 seedlings of salt tolerant lines (LPT 144 and 171) growing in water with 0.5% NaCI added. aBoth lines show severe symptoms (Fig. IA), but line no. 171 recovers and produces a growth s~milar to control, while line no. 144 recovers partly (Fig. IB).

Sun Z, Zhao C, Zheng K, Qi X, Fu Y (1983) Appl Gene 67: 67-73 Tal M (1984) In: Staples RC, Toenniessen GH (eds) Salinity Tolerance in Plants, John Wiley and Sons, New York Chichester Brisbane Toronto Singapore, pp 301-320 White PR (1943) A Handbook of Plant Tissue Culture, Ronald Press, New York Wong CK, Woo SC, Ko SW (1986) Bot Bull Academia Sinica 27: 11-23

ACKNOWL~ This work was funded by USAID Grant no. 936-5542-GSS-3037-00. The authors would like to express their appreciation to Professor Murray W. Nabors for his kind advice and collaboration; the Rice Division, D e p a r t m e n t of Agriculture, for supplying the materials used in this work and the Office of Research Affairs, Chulalongkorn University, for its support.

Yamada Y, Ogawa M, Yano S (1983) In: Cell and T i s s u e C u l t u r e Techniques for Cereal Crop Improvement, Science Press, Beijing, pp 229-235

RF~CES

Yoshida S, Ogawa M, Suenaga K, Ye HC (1983) In: Cell and Tissue Culture Techniques for Cereal Crop Improvement, Science Press, Beijing, pp 237254

Bentley M (1959) Commercial hydroponics. Books, Johannesburg.

Bendon

Y e o AR, F l o w e r s TJ (1984) In: Staples RC, Toenniessen GH (ads) Salinity Tolerance in Plants John Wiley and Sons, New York Chichester Brisbane Toronto Singapore, pp 151-170