Cotton is an important economic and fiber crop. To study the effects of salt stress on cotton have profound significance for breeding of salt resistant varieties. The study of the effect of salt stress on cotton growth and development with the methods of irrigation cotton field using sea water or salt water or using salt pond have been reported. But these methods have many abuses, such as longer study cycle, higher study cost, more work time and unsteady because that the results were influenced easily by around environment. So, we must investigate new methods to evaluate exactly the effect of salt stress on cotton growth and development.
With the development of modern biotechnology, people are attempting to study the influence of salt stress on plant growth and development with plant tissue culture technique and certain progress (Zhou et al., 1989) have been obtained.
Recently, great progress has been made in cotton tissue culture (Zhang and Feng, 1992a, b) and the procedure of cotton somatic embryogenesis and plant regeneration has been establishment (Trolinder and Goodin, 1987; Voo et al., 1991; Zhang and Li, 1992). These would lay a foundation for the use of plant tissue and cell culture technique on cotton breeding, production, preservation and appraisal of germplasm (Zhang and Feng, 1992b). The study of the effects of salt stress on cotton tissue culture was the direct application on the study of cotton salt resistance. Only by clearing the effects of salt stress on the induction, growth, proliferation, differentiation of callus and plant regeneration of cotton tissue culture, can we go deep into studying the screening of salt-tolerant variation and rapidly appraisal in vitro of cotton. So some articles about the effect of salt stress on cotton tissue culture have been reported (Li et al., 1992; Wang et al., 1991), but only the effect of salt stress on the induction of cotton callus were discussed. The other questions, such as the effect of salt stress on the proliferation, differentiation, embryogenesis of callus and plant recovery have not been reported. The purpose of this experiment is to look for a method of evaluating cotton salt resistance rapidly and exactly.
Materials and Methods
Gossypium hirsutum L. cv. Coker 201 was chosen for this study because it could be induced somatic embryos and plant regeneration easily. Seeds that were delinted with 95 percent H2S04, were steriled in 0.1 percent HgCl2 for 10 minutes, followed by three rinses in sterile distilled water and than soaked in sterile water for 3 to 7 hours. Softened seed coats were removed and the kernels were germinated for 3 days on MS basal medium at 282 under darkness. Transversal hypocotyl sections ("discs") ranged from 3- to 5-mm thickness were take from the lower half of 3-day seedling and were inoculated on MS basal medium supplemented with 0, 3, 5, 10, 15, 20, 25, 30 g/L NaCI respectively to study the effects of various amount of salt stress on the induction of callus. The callus that were induced on MS medium without NaCI were proliferated by subculture every one month on MS basal medium supplemented with 0.1 mg/L KT, 0.2 mg/L IAA and 0.1 mg/L 2, 4-D, which were prepared to use in late experiments.
After 30 days, callus that were subcultured on MS medium without NaCI were transferred to the MS medium supplemented with 0, 1, 3, 5, 7, 10, 15, 20, 30, 40 g/L NaCI respectively to study the effects of salt stress on the growth and proliferation of callus, somatic embryogenesis and plant regeneration in cotton tissue culture. The growth rate of callus that explant produced was expressed by the follow methods: +++,++, + stand for respectively good, moderate, poor. The growth rate of callus was used to compare callus response to salt stress, it was expressed by the follow formula:
All experiments were carried out in a controlled environment growth room at 291, irradiance of 2000Lux or so, 14 hours light/10 hours dark photo periods.
Results and Discussion
Effect of NaCI stress on the induction of cotton callus: NaCI deferred the appearance and reduced the induction rate of cotton callus (Table 1).
|Table 1:|| Effects of NaCI stress on the induction of cotton callus
|Table 2:|| Effects of NaCI stress on the proliferation of cotton callus
|Table 3:|| Effects of NaCI stress on the differentiation and development of cotton somatic embryos
|Table 4:|| Effects of NaCI stress on the germination of cotton somatic embryos and plant regeneration
Callus appeared on MS medium without NaCI in 6 days and the percentages of callus was 100 percent after 40 days of culture. The percentage of inducted callus decreased with the increasing of NaCI concentrations. When the NaCI level increased to 30 g/L, the explant were not able to produce callus. High NaCI levels resulted in the death of hypocotyl explant, which the semilethal concentration was 15 g/L and the lethal concentration was 30 g/L. The colour and growth rate of callus produced on MS medium with various NaCI levels were different. The colour transformed from greyish brown to deep yellow, the growth speed declined along with the increasing of NaCI levels.
Effect of NaCI stress on the proliferation of cotton callus: NaCI had a great effects on the growth and proliferation of cotton callus (Table 2). The growth was reduced with the increasing of NaCI levels, but the effects were different among various NaCI level. Low concentration (5 g/L) of NaCI affected little survival of callus, but the growth of the treatment with O g/L of NaCI is almost 2.5 times then that of the treatment with 5 g/L of NaCI; moderate concentration (10-20 g/L) of NaCI inhabited the growth of callus and high concentration (20 g/L) of NaCI stopped the growth of callus. Callus or cell morphology differed with the various concentration of NaCI. With the increasing of NaCI level, the moisture content of callus decreased and the ratio of dry weight to fresh weight increased. Cytology observation indicated that the cells lessened and cytoplasm densened with the increasing of NaCI concentrations.
Effects of NaCI stress on the differentiation and development of cotton somatic embryos: Cotton somatic embryogenesis was inhibited by NaCI (Table 3) and a clear relation between the levels of NaCI added to the medium and somatic embryogenesis event was observed. 1 g callus that were cultured on MS medium without NaCI could produce 228 somatic embryos, but that were cultured on MS medium supplemented with 10 g/L NaCI could only produce 90 somatic embryos. When the NaCI concentration was more than 30 g/L in the MS medium, embryogenesis could not take place from the callus.
Salt stress affected not only the total number of the embryos produced, but also the developing course of somatic embryos and shus it affected the ratio of immature to mature embryos. With the increasing of NaCI concentrations the percentage of the mature embryos decreased and that the immature embryos increased The callus could not produce somatic embryos at 30 g/L NaCI. The somatic embryos that were produced on MS medium supplemented with 0-10 g/L NaCI could develop to cotyledonary stage, but we could only observe torpedo embryos on the MS medium supplemented with 15-20 g/L NaCI. On other medium with higher concentration of NaCI, we could not observed somatic embryos at any development stage.
Effects of NaCI stress on the germination of cotton somatic embryos and plant regeneration: Somatic embryos that were produced from the MS medium supplemented with various concentration NaCI stress were transferred to the germinated medium (Zhang et al., 1993a) without NaCI and germinated soon. Somatic embryos germination begun on the medium with either root initiation or shoot growth within 10 days.
The effects of stress caused by various NaCI concentration on germination of cotton somatic embryos and plant regeneration were various (Table 4). The percentages of embryos that developed shoots, roots and both shoots and roots were inhibited by adding NaCI to MS basal medium. There after, the percentage of germination or plant regeneration of cotton somatic embryos decreased with increasing NaCI concentrations.
Regenerative plants were also affected by NaCI that was added to MS basal medium. The change of the percentage of normal seedling with different NaCI concentrations was the main expression. Along with the increasing of NaCI level, the percentage of normal seedling decreased, the percentage of abnormal seedling increased. When the concentration of NaCI was up to 15 g/L, there were not any normal plants in regenerative plants.
On this basis, we have been studying to screen salt-tolerant variation and we have obtained salt-tolerant embryogenic cell lines and we have obtained somatic embryos and regenerative plants from these cell lines (Zhang et al., 1993b, 1995).
We thank Mr. Yang Weihua for correcting for the English language of this paper.