The day by day increase in human population is increasing the food demands which intrun needs the employment of barren lands for cultivation. A large bulk of such land suffers from high salt. The energy requirements and other factors involved in reclamation of lands are not economic. Hence, evaluation of plants resistance to these stressed soils is the solution to the problem. There are classical ways for breeding plants resistance to salt stress but these are lengthy, time consuming and source oriented. Novel biological techniques like tissue culture can be much useful in this regard because they are economical, shortcut and easy to handle. The study being reported deals with the application of tissue culture for induction and screening of salt tolerance in Pakistani rice varieties Basmati-370, KS-282 and IR-6. For this purpose sterilization techniques were monitored, chlorox (a commercial NaOC1 preparation was preferred over other sterilants. Among the nodal protein , root tips and mature seeds, the seed proved to be the optimal explants source for callus induction and subsequent regeneration of the plants, 28 Co was worked out---- be the best temperature for callus induction and callus multiplication using the seed explant. For organogenesis 26 Co was the optimal temperature. Light did not play any significant role in callus induction, Callus formation frequency was maximum at a pH range of 5.5 to 7.0 yeast extract helped in callus induction when used in 0.5 % concentration , callus induction frequency was the highest in the sucrose range 2-4 % .High amounts of sucrose even proved to be lethal. Effect of glucose in this regard was more severe. The effects of four different auxins i.e, IAA (indole-3-acetic acid) IBA (indole-3-butyric acid) NAA naphtanlene( acetic acid) and 2,4-D (2,4-dichloropthnoxy acetic acid) on callus induction were monitored, 2,4-D was found to be the best for callus induction at al level of 2 mg/1. If a cytokine were added at low concentration in the callus induction and maintenance media, long term callus regeneration is increased. The highest regeneration was obtained at a combination of BA (6-benzy1) (aminopurine) and NAA. Sodium chloride, calcium chloride and sodium sulphate caused growth arrest and decrease in cell multiplication , 1% individual salt was used for cell screening but shoot regeneration was achieved at 0.5 % of the salt. About 100 plants were selected in this way. These were potted and twelve survived. The plant progeny of only one variant was tested for slat tolerance and for other characteristics, Cytological studies of the metaphase I of this plant were also conducted. In total 300 cell were observed . Out of these 50% were aneuploids, 23 % PMCs with 10(II) and 33% with 11(II), remaining 43% contained the normal numbetr of chromosomes. No other association was found in this material. It is hypotheised that 2, 4-D and Kinetin 96-furfruy1 aminopurine) interaction enhances DNA replication; the DNA polymerases cannot complete the Job of proof reading. The SOS repair system operates and mutation is even more enhanced in the presence of NaC1. The intrun gives rise to ploidy change . Seeds form 9 different panicles were sown for germination at five different ECe levels to detective the tolerance limits. IR-6 and Nona Bokra were used as sensitive and control. Germination and morality data were recorded for 8 counts; all counts were taken five day after showing statistical analysis showed a significant difference in germination and morality rates at different times and with different seeds. One plant was matured at ECe=15 and tow matured at ECe=10 analysis for Na, K, Fe,Mn, Zn, and Cu revealed Na exclusion at least in the plant growing at 15 mmh/cm.. This study revels that tissues culture can generate variability in crop plants. This has a lot of potential for crop improvement, especially for cereals, Al though salt tolerance is a multigenic character is was introduced in the rice plants. Selection was also made in vivo. This technique can be more useful in selection against biotic stresses because they are controlled by single genes. This techniques can also be useful for the improvement of agronomic traits in rice
Physicochemical analysis of the brown rice of the above plants was also carried out. According to different quality parameters our plant is quite comparable to our indigenous varieties. Chemical analysis reveals that the crude protein is 8.90 % in our mutant while it is 9.97 % in IR-6 , 9.13% in Nona Bokra, 8.9 % in Basmati -370 and 8.39% in KS-282. Glutrmic acid which is the main amino-acid of these five varieties is dropped in our mutant from 1.29 to 0.97 g/100g. An increase in praline was also noted. Lysine content was the highest in cases of Nona Bkora and KS-282 followed by Basmati-370, mutant and IR-6. Methionine content increased from 0.11 to 0.18 the. The fat and fatty-acid compositions were also investiogated. The fat content was comparable with mother variety . The main fatty acids of all the varieties are palmitic, oletic and linoleic acids. Other fatty acids detected were myristic, steraic, linoleic , arachidic, gedoleic, eicosadienoic, behenic, enucic and docosadienoic acid. Myristic acid and docosadienoic acids were detected only in the mutant and Nona Bokra rice grains. However, the fatty acid composition was performed for four varieties. The maximum amount of Na was observed in Basmati-370 followed by IR-6, Nona Bokra and the mutant. The highest amount of K was detected in Basmati-370 and the best was in IR-6. Cu, Zn, Fe and Mn contents of all the four cultures are also reported. Crude fibre was highest in Basmati-370 followed by IR-6, KS-282 and the mutant and Nona Bokra. Chemical analysis indicates that myristic acid and docosadienoic acid may have some importance in salt tolerance
Since anther culture is a short-cut method for varietal evaluation, experiments were conducted to establish the conditions for callus induction. Three different basic media were employed, 8P, N6 and potato medium. Over 100 cross combinations of these media were tested. The effect of spinning the anthers was also investigated. The anthers were inoculated into the media either after chilling or without chilling. The effect of spinning was found to be unfavourable in all cases. No callus or swelling was observed the Basmati-370. No true callus was found in IR-6 but initial swelling was observed in combinations containing(I) 8P medium 2mg/1 2,4-D and 65 sucrose and (II) 8P medium alongwith 4mg/1 2,4-D and 3% Sucrose, call were obtained from KS-282 anthers in four combinations which were modification of 8 p medium . The optimal percentage of sugar was 3 % while of the auxin was 47 mg/I, 2, 4-D. More detailed studies are needed on this aspect.
A Colour pattern of green, xantha, tigraina, viridus, in callus borne shoots, seedling from seed in virto or the first head progen, of the green plants raised through callus culture. The colour variation was in some cases temporary and in other cases permanent. Sucrose cause colour variation in rice seedlings. Glucose also induced colour changes leading to viridus formation. These changes were reversible. Additional of kinetin lowered the colour variant seedling formation caused by high concentrations of sucrose. The basal medium as a whole also brought also brought about some colour changes. Auxin treatment increase colour variant formation and it was higher in cases of 2, 4-D than in case of NAA. The seedlings did not revert to green colour on any medium. Most of the colour variant were albino. Colour variants were also observed in NaCl, alone or with kinetin However, the frequency was lowered was lowered by the presence of the kinetin.
The colour variant to green plant ratio was increased by increasing the incubation time of the call in auxin medium. Higher temperatures also caused significant rise in colour variation among the callus regenerants. Three types of chlorophyll variations were observed in the first head progeny of three Basmti somaclones of rice. These included albino, viridus and striata. These results cab be sued to avoid or at least lessen the albino rice regenerants. The results may reveal some new dimensions of chlorophy 11 genes.
The effect s of Nicolinic acid, try ptophang indole acetic acid (IAA) and serine, in combination with kinetin (K) and alone, were investigated on embryogenic callus (E Callus) induction in Basmati-370, IR-6 and KS-282 rice varieties. There was no induction frequency in any of the three rice varieties under study. However, callus induction frequency was lower in Basmati-370 than in the other two varieties. Embryogenic callus induction frequency was statistically higher in the case of nicotinic acid (1.6mg/I) combined with kinetic, in all the three varieties. In Basmati-370 embryogenic callus induction was also lower than the two other varieties.
Nicotinic acid, IAA, tryptophang and serine showed no positive effects on embryogenic callus induction when used in medium without kinetin. It was observed that tryptophang and IAA do not enhance the ( E Callus) induction frequency. However, nicotinic acid has an additive effect with kinetin in E callus formation in these rice varieties. Thus, a cytokinin must be added alongwith nicotinic acid to media employed for callus induction and muhiplication to get embryogenic calli at maximal frequency. This intrun will enhance the number of plants regenerated from the calli when transferred on regeneration medium.