About 30% of the world's rice is grown in rainfed, lowland regions which are often subjected to drought at least once during their growth. For planting in these drought-prone regions, the past approach has been to use traditional plant breeding techniques utilising existing variation in other cultivars or wild relatives. However, the efficiency of these practices is low, due to the long term selection and backcrossing programs needed. Somaclonal variation, the rapid production of genetic variation generated through a tissue culture cycle, has been shown to be useful in many plant breeding programs including rice and could be expected to generate novel variation for drought tolerance. Thus, the objectives of the present study were to; a) improve upon reported callus culture techniques to gain high frequency plantlet regeneration of a range of southeast Asian rice cultivars and b) assess somaclonal variation among the selfed progeny with emphasis on their reaction to drought stress and other agronomic characters. Some of the somaclones originated from cultures which had been challenged with an osmotic stress agent, polyethylene glycol. Wide hybridisation was also attempted in order to introduce genes from wild relatives with known drought tolerance to cultivated rice.
Plants were regenerated from seed callus cultures, using a four-step culture system. Overall, optimal conditions for regeneration were: callus induction on Murashige and Skoog's nutrients containing 2,4-dichlorophenoxyacetic acid (10 µM), sucrose (59 m̄M), casein hydrolysate (3 g l-1) and agar (8 g l-1), callus proliferation on a Murashige and Skoog's nutrients with 2,4-dichlorophenoxyacetic acid (5 µM), sucrose (39 mM), casein hydrolysate (3 g l-1) and agar (8 g l-1); shoot and root induction on Murashige and Skoog's nutrients plus sucrose (59 mM), casein hydrolysate (3 g l-1) and agar (8 g l-1) alone or supplemented with 1-aminocyclopropane-1-carboxylic acid (5 µM) and kinetin (5 µM), and plantlet development on 0.5 strength Murashige and Skoog's nutrients plus sucrose (59 mM), casein hydrolysate (3 g l-1) and agar (8 g l-1) without plant growth regulators. A single regeneration medium for all cultivars could not be developed because of genotypic differences. In all, 788 regenerants from callus culture were produced and transferred to soil, of which 56% survived to set seeds.
In vitro selection in liquid culture was attempted to mimic osmotic stress in cell cultures. However, no correlation was found between cellular growth response to polyethylene glycol and published drought scores for the cultivars studied. In vitro selection involving polyethylene glycol was, however, useful in enhancing plantlet regeneration in one cultivar, Nam Sagui 19. In all, 201 regenerants from suspension culture were produced and transplanted to soil, of which 95% survived to set seed.
Emphasis was placed on plant character variation in the selfed progeny coming from the secondary regenerant plants (R2 generation). Replicated glasshouse trials were performed with selfed progeny regenerants of 45 Calrose 76, 33 Khao Dawk Mali 105, 13 Kurkaruppan and 50 FR13A families from callus culture; and 23 Nam Sagui 19 and 20 Khao Dawk Mali 105 families from suspension culture; plus parental families, a total of 2208 plants. Variant phenotypes were observed with segregation for albinism, altered morphology and days to flower. Variant families, with higher or lower values than parents, were observed for days to flower in Nam Sagui 19 and Khao Dawk Mali 105 and with grain yield in Khao Dawk Mali 105. However, only one Khao Dawk Mali 105 family significantly outperformed the parents with respect to grain yield. In all, 91% Nam Sagui 19 families varied for one or more characters as compared to the parent followed by Calrose 76 and Kurkaruppan (84% each), FR13A (58%) and Khao Dawk Mali 105 (50%). In most cases, the variation observed could be explained by single recessive gene mutations. Segregation data suggested that changes in minor genes caused variation in days to flower, seed weight and plant height in some families. Chromosome counts revealed that a number of autotetraploid families in cultivars Calrose 76 and Nam Sagui 19 had been developed, otherwise, all Calrose 76 somaclones were diploid. These tetraploid families had larger leaves, stems and seeds with reduced fertility and grain yield.
Preliminary experiments to develop a glasshouse drought tolerance test and to characterise the drought tolerance of parental cultivars, showed that differences among cultivars could be determined with high repeatability when shoot dry weight was used as the measure of drought tolerance. In the best test developed, the soil water level was allowed to fall until signs of plant wilting were observed after which the soil water level was returned to field capacity This cycle was repeated a further seven times before measurable differences could be obtained between drought susceptible and drought tolerant plants.
Glasshouse trials were performed to assess the reaction to drought of 34 Kurkaruppan, 34 Calrose 76, 33 FR13A and 19 Khao Dawk Mali 105 R2 families derived from callus cultures: and 19 Khao Dawk Mali 105 families and 10 Nam Sagui 19 families derived from suspension cultures. Of these selfed regenerant progeny, three (2%) families were identified as having a significantly improved drought tolerance compared to their parental controls. In vitro selection with polyethylene glycol gave rise to one Khao Dawk Mali 105 (7%) family with increased drought tolerance with no improvements observed in selfed progeny families derived from suspension culture without polyethylene glycol.
These results indicate that somaclonal variation for increased tolerance to environmental stresses can be generated in tissue culture and may be selected for under appropriate glasshouse conditions. However, not all of the three drought tolerant variants from this study performed well in regard to agronomic traits as only one FR13A selfed progeny family performed as well as the parent in all other traits measured.
Wide hybridisation involving crosses between O. sativa and either wild or hybrid rice was attempted. Using an embryo rescue technique, although 259 embryos were formed, all were lost during this step.
It is evident that significant genetic variation can be generated in O. sativa using a tissue culture cycle. Some of these demonstrated variants, such as those with increased drought tolerance, increased yield, altered phenology and altered growth habit have potential use in rice breeding programs.