In vitro screening and molecular genetic markers associated with salt tolerance in maize

Salinity is one of the major limitations of plant growth and productivity all over the world. In vitro screening with marker assisted selection can greatly facilitate the selection and isolation of useful tolerant lines. The genetic basis of salt tolerance was examined in salt tolerant (EC 558706), moderately tolerant (EC 558719) and salt sensitive (EC 558620) maize genotypes. Callus lines from mature embryos were developed and exposed to culture media containing, 0.5, 1, 1.5 and 2% NaCl concentrations. The treated calli were used to detect DNA based molecular markers associated with salt tolerance by random amplification of polymorphic DNA (RAPD) technique. Ten (10) polymorphic bands were found related to salt tolerance in maize. The analysis revealed A 11 (1200, 600, 300 bp), C 20 (300 bp), OPA 10 (2800 bp), OPA 13 (900, 600, 250 bp), OPX 11 (800 bp), OPI 01 (700 bp) and OPK 20 (700 bp) primers associated with salt tolerance which can be utilized in breeding programme via ., marker assisted selection and developing salt tolerant genotype by genetic transformation.


INTRODUCTION
Cereal culture is of great economic importance in an agricultural country like India (Zair, 2003).Maize (Zea mays L.) is the most important cereal crop of world agricultural economy.Among the cereals and grain crops, maize rank third in production in the world being suppressed only by rice and wheat.
Salinity is one of the major limitations of plant growth and productivity all over the world (Flowers, 2004;Foolad, 2004).Maize is moderately sensitive to salinity and considered as the most salt sensitive of the cereals (Maas and Hoffman, 1977).In vitro selection shortens the time considerably for selection of desirable trait under selection pressure without having any environmental influence (Bressam et al., 1985;Rosas et al., 2003).Testing genetic resources for productivity, quality parameters and stress tolerance, field trials are usually time consuming.Molecular variation in tissue culture derived plants has been characterized at DNA level (Jain, 2001).
So, the modern one in which natural genetic variation is considered and selection in stressful environment is carried out with molecular marker technology at genetic level is method of choice (Karp et al., 1993).
Various molecular markers viz., randomly amplified polymorphic DNA (RAPD) restriction fragment length polymorphism (RFLP), amplified fragment length polymorphisms (AFLP), inter-simple sequence repeat (ISSR) and simple sequence repeat SSR are being used to characterize the induced genetic variation.Amongst these molecular markers, RAPD analysis is a simple, quick, easy to perform and require small amount of DNA (Welsh and McClelland, 1990;Williams et al., 1991).
These benefits justify the frequent application of RAPD technique in genetic variability studies (Mondal and Chand, 2002;Bennici et al., 2003;Feuser et al., 2003).Keeping these considerations in view, the present work was aimed at in vitro screening for salt tolerance and to *Corresponding author.ashu.raj123@gmail.com.Tel: +91 9975051209.

Callus induction
The mature seeds were surface sterilized with 70% ethanol for 2 min and 0.1% mercuric chloride for 15 min.The seeds were soaked in 2,4-dichlorophenoxyacetic acid (2,4-D) (4 mg/l) and swollen mature embryos were removed after 72 h.The plumule and radicle section (2.5 mm) was longitudinally sliced into halves and inoculated on MS basal medium supplemented with 2,4-D.

DNA isolation and PCR amplification
Genomic DNA was extracted from 30 days old salt treated calli (100 mg) by cetyl trimethyl ammonium bromide (CTAB) method (Saghai- Maroof et al., 1984) with some modifications; the concentration of some chemicals was standardized to get maximum quantity of genomic DNA.
Twelve (12) out of 33 random decamer primers from different sets (A, RC, C, O, Z, OPA, OPC, OPE, OPG, OPH, OPI, OPM, OPR and OPX) of Operon Technology Inc, USA were chosen on the basis of reproducible and scorable amplification products for salinity specific gene expression studies (Table 1).
A 25 μl mixture contained 20 ng of genomic DNA, 1U Taq DNA polymerase (Genetix), 1XPCR buffer, 4 mM MgCl 2 , 0.2 mM of dNTPs (Genetix), and 30 pmol primers.Amplifications were carried out using a 96 thermal cycler (Bio metra) programmed for 40 cycles as follows: initial denaturation at 94°C for 5 min, further denaturation at 94°C for 1 min, annealing at 36°C for 1 min, extension at 72°C for 1 min and final extension for 8 min at 72°C.The PCR products were resolved at 100 V for 4 hon 1.2% agarose gel prepared in 1x Tris-borate-EDTA (TBE) buffer.Gel was photographed using Gel-Documentation system (ALPHAIMAGER TM 2200).

Callus induction
Swelling of the scutellum region and the appearance of small calli (0.5 to 1 mm diameter) from mature embryos were observed after 10 days.The callus induction in terms of growth was relatively low (Figure 2) at a low level of 2,4-D (2 mg/l), however maximum induction was observed at 4 mg/l 2,4-D.Higher concentration of 2,4-D (5 mg/l) did not significantly change the callus quantity or quality.Hence, it was found that 4 mg/l 2,4-D is optimal concentration for callus induction.
The optimised concentration of 2,4-D (4mg/l) was used in salinity treatments.The salt treatment was measured in terms of callus growth.Callus cultures on salt selection media showed decrease in callus growth with increase in salt concentration (Table 2).

RAPD markers associated with salt stress
The gene expression under salt stress among the selected genotypes was analysed using RAPD markers.Out of the 33 primers, only 12 primers were amplified and developed a total of 333 bands.The lowest number of bands ( 19) was developed by primer OPK 20, while the highest (45) was developed by OPA 10.Seven primers viz., A 11, C 20, OPA 10, OPA 13, OPX 11, OPI 01 and OPK 20 showed unique bands (Table 3 and Figure 1).A positive marker is a band generated in salt treated calli whereas, negative marker is a band obtained in control.The positive unique bands obtained in salt tolerant calli  will be used as a RAPD marker for developing salt tolerant genotypes.

DISCUSSION
The determination of absolute tolerance under in vitro conditions has rather been impossible because of the complex interactions between plant systems and the environmental factors.However, the development of improved stress tolerant variety appears to be promising.
The in vitro techniques have become useful in generating novel genetic variability in many seeds as  well as vegetatively propagated plants (Maluszynski et al., 1996).
In the present study, the frequency of callus formation from mature embryos was low (Figure 2).This follows results reported by Wang (1987) in maize.The role of plant growth regulators in cereal tissue culture has been reviewed by Bhaskaran and Smith (1990).In general, auxins usually 2,4-D in the range of 1 to 3 mg/l, are essential for the establishment of embryogenic callus from cereals embryos.The use of 4 mg/l 2,4-D to induce callus formation from maize embryos is a critical factor.The result of this study suggests that it might be possible to improve regeneration from mature embryos by optimizing the compositions of regeneration media for specific genotype.Hung and Wei (2004) obtained 90% frequency of primary calli from mature embryo of maize at 4 mg/l 2,4-D.
Six positive markers viz., A11, C 20, OPA10, OPA 13, OPI 01, OPK 20 were found to be related with salt stress which are in conformity with the results obtained by the Zacchini et al. (1997) (1997,1998) detected one RAPD marker for salt tolerance and three for drought tolerance in maize while they developed two positive and two negative molecular markers for salt tolerance in maize (Abdel-Tawab et al., 2001) using bulk segregation analysis.
These results suggest that this polymorphism could not be caused by selection of pre-existing variants in the zygotic embryos population from which the calli were derived, but must be attributed to somaclonal variation occurring during callus formation in response to salt stress.
The results from the present study are in agreement with the detection of genetic variation in response to salt stress with RAPD analysis by Rasheed et al. (2001) and Patade et al. (2005).Rasheed et al. (2001) analyzed genetic variability among salt stress (200 mM NaCl) calli of sugarcane and potato using RAPD technique.Out of six primers used, only two primers enabled the identification of polymorphism among salt tolerant plants of sugarcane whereas Patade et al. (2005) obtained a unique band of 500 bp in salt tolerant regenerated lines of sugarcane.
The RAPD technique has proved very sensitive for genetic variation studies and fastest method for detecting a great number of genomic markers in in vitro selected salinity tolerant calli in a short period of time.

Conclusion
In vitro selection shortens the time considerably for selection of desirable trait under selection pressure and has been used for selection of abiotic stresses while the RAPD technique has proved very sensitive for genetic variation studies and fastest method for detecting a great number of genomic markers in in vitro selected salinity tolerant calli in a short period of time.

Figure 1 .
Figure 1.RAPD profile of salt treated genotypes with primer A11.

Table 1 .
List of primers along with their sequence used for RAPD analysis.

Table 2 .
Effect of salt stress on growth of callus culture.
*Weight taken after 25 days of inoculation on different salt concentration medium.

Table 3 .
Characteristics of the amplified products obtained by RAPD primers.