Callus formation and somatic embryogenesis in sugarcane ( Saccharum spp L . ) using various concentrations of 2 , 4-D and RAPD analysis of regenerated plants

The present study was carried out to build up an efficient protocol for various concentrations of 2, 4D for “Callus formation and somatic embryogenesis in sugarcane (Saccharum spp L.) using various concentrations of 2, 4-D and RAPD analysis of regenerated plants in the laboratory of Biotechnology Nuclear Institute of Agriculture, Tandojam during the year 2015 to 2016. Three sugarcane varieties Bl4, NIA-2010, NIA-2011 with different concentrations of 2, 4-D (0.5.1.0, 2.0, 3.0 and 4.0 mg L) were used respectively , while 3.0 mg LKin+IAA+IBA was used for callus proliferation and shoot formations. Four different concentration of IBA were used for rooting purposes 0.5, 1.0, 2.0 and 3.0 mgL+20 g Sugar. Embryogenic callus was obtained by culturing young apical meristem. Three sugarcane clones BL4, NIA-2010 and NIA-2 011 were developed in the field area for eight month and sand was used for tissue culture somatic embryonic callus study. Apical meristematic region was used for callus formation and somatic embryogenesis induction on 0.5, 1.0, 2.0, 3.0 and 4.0 mg L.2, 4-D actively growing callus was subcultured on kin. IAA, IBA, 3.0 mg L. Maximum callus proliferation and number of plantlets shoot length and regeneration growth were observed in plants that 1.0 and 2.0 mg L2, 4 -D callus was taken from. Maximum chlorophyll mutation frequency was observed in NIA-2010 and BL4 grown on 1.0 mg L 2, 4-D. Maximum number of roots were observed in BL4 when 1.0 mg LIBA+ 20% sugar was applied; variability was also obtained through callus culture and confirmed through random amplified polymorphic DNA (RAPD) techniques.


Introduction
Sugarcane (Saccharum spp L.) hybrid is a profitable crop cultivated in 74 countries around the globe [1].It is a perennial crop and belongs to Poaceae family, which contributes approximately 75% of worldwide sugar production [2].Other than sucrose production, it is likewise utilized as part of Ayurvedic drugs and for the treatment of skin and urinary tract diseases, bronchitis, heart conditions, loss of milk production, cough, anemia, constipation and also general debility [3].Another essential utilization of sugarcane is the production of press mud, which is utilized as a source of organic matter and supplements for crop production [4].Sugarcane is the world largest crop by production, the annual production of sugarcane in the world is 1.83 billion tons having an area of 26.1 million hectares.The average yield of sugarcane in the world is 70.24 tons/ha.Pakistan produces 58.4 million tons by harvesting 1.04 million hectare and the yield is 55.8 tons/ha [5].In Pakistan, sugarcane is the biggest industrial cash crop second to cotton and is basic as a result of its huge share in agriculture sector.Various demands for sugarcane in agricultural industry has vitalized the need for continuous varietal improvement and extensive research on the crop [6] An extensive breeding work, management and cultural practices are required to narrow down this big gap.Although, a good crop is harvested in Pakistan, it does not flower and produce seed (fuzz) under normal growing conditions in the country.Non-sporadic flowering and natural viable and fertile seed production has usually been a problem in Pakistan.Modern sugarcane varieties have multifarious genetic characteristics and low fertility that renders their genetic improvement is very difficult through traditional breeding.Thus the substitute method for propagation is in vitro culture techniques and induced transformation being utilized to make the new genetic variability for the determination of desired clones of sugarcane [7].In Optimized conditions for callus induction in sugarcane have been studied investigated in the present study.This optimized protocol will help in establishing efficient method for the genetic transformation in callus of the important sugarcane varieties for resistance to various stresses.Present study was conducted to induce the callus and somatic embryogenesis in different genotypes of sugarcane (BL4, NIA-2010 and NIA-2011) with the use of different concentrations of 2, 4-D (0.5, 1.0, 2.0, 3.0 and 4.0 mg L -1 ).Embryonic calliis then regenerated and analyzed through DNA RAPD marker for genomic variations among the somaclones and its mother plant.

Materials and methods
The research was carried out at Nuclear Institute of Agriculture,Tandojam for callus formation and somatic embryogenesis in sugarcane (Saccharum sppL.) using various concentrations of 2, 4-D and RAPD investigation of regenerated plants of BL4, NIA-2010 and NIA-2011.Laboratory used was well equipped and facilitated by culture and washing room, laminar airflow cabinet, culture vessels, incubation room, autoclave, pH meter, electric oven, glass house, centrifuge machine, spectrophotometer, PCR and Gel documentation and miscellaneous needs.

Preparation of micronutrient stock solution
The quantities each of salts (Table 1) were measured and transferred into the conical flask and sterilized (ddH2O), distilled water 700ml was then delivered into conical flask.Solution was stirred to dissolve properly and transferred to volumetric flask (1L) and made up to volume.The flask was labeled and stored in the refrigerator.

Preparation of vitamin based stock solution
The quantities of ingredients for the vitamin stock presented (Table 1) were measured into conical flask having some quantity of sterilized distilled water and mixed thoroughly using magnetic stirrer.The solution was transferred into volumetric flask 1l and made up to volume and the flask labeled and stored in refrigerator till use.
The following are the growth regulators stock solution for somatic embryogenesis callus formation shoot regeneration and rooting forming culture media: a) 2, 4-Dichloro phenoxy acetic acid; b) Kinetin; c) Indol-3-acetic acid and d) IBA Indole-3butyric acid.

Preparation of laminar air flow cabinet
The laminar air flow cabinet was switched on for 30 min.Before starting the process of culturing, it was uncontaminated with absolute alcohol like 70% ethanol.After laminar air flow cabinet was further sterilized with all required materials and kept inside the cabinet.

Selection of explants, surface sterilization and culture
Ten (10) explants containing apical meristems were taken from every genotype.Superfluous part of cane top was uprooted and the remaining initially washed with refined water and afterward disinfected with 70% Alcohol (ethanol) for 1 min and 20% sodium hypochloride solution for 20 min.
After sanitization explants material were washed with double refined water 2 to 3 times to uproot any hints of disinfectant under aseptic conditions in laminar air flow cabinet.These sterilized meristem or shoot tip was cut or isolated from stem and a Vshaped cut with sterilized knife applied.In the meristem tip culture, the cut was a small piece applied 0.3 to 0.5 mm below the tip of the dome and the excised tissue (explants) was removed along portion of procambial tissue and immediately planted on media.

Molecular marker RAPD analysis of somatic embryo-derived plants among mother plants
RAPD is any DNA Segment that is amplified using short oligodeoxy nucleotide primers of arbitrary nucleotide sequence and polymerase chain reaction procedure.

Buffer solution genomic DNA extraction
Total genomic DNA was separated from samples of young leaf tissue of all the three varieties Bl4, NIA-2010, NIA-2011; three specimens of that recovered from somatic embryo callus were taken from field plantlets' and determined through nodal shoot multiplication and somatic embryogenesis from every accession.Leaf tissues were ground into fine powder in liquid nitrogen using a mortar and pestle.Buffer chemicals for 500X reactions used are: 50 ml Tris-HCl, 140 ml MATAB, 20 ml EDTA, 10 g NaCl, 5 g PEG 6000 and 2.5 g Na2SO3 (Table 2).Cycler System and initiated by a denaturation of 3 min at 95°C followed by 40 cycles of 94°C for 1 min, 37°C for 1 min, 72°C for 2 min.The amplification was completed with 1 cycle of 7 min at 72°C.

Results and discussion
The results on weight of explants/bottle, weight of Callus/bottle, weight of proliferation /bottle, type of callus, number of shoots per bottle, shoot length per bottle, number of chlorophyll mutants per bottle and number of roots of sugarcane callus induction under various concentrations of 2, 4-D recorded are presented in Tables 1, 3 to 8 and their analysis of variance and data shown in Table 9.

Types of callus (A) regenerable and (B) non-regenerable
Study of callus type is also important for the identification of various responses of callus tissues.Two types of callus were discovered (and) signified in the study, as re-generable callus Type-A and non-regenerable callus Type-B.The regenerable callus were distinguished and perceived as dry nodular compact and light yellowish in color as wild, however, B type callus was watery, translucent and non-nodular in shape (Figures 1 and 2).Whereas, Type A (dry nodular and compact callus) was found in clones of BL4, though, NIA-2010 andNIA -2011 had smooth conservative callus denoted as Type A and B (Table 1).The outcomes are upheld by Seema et al.Maximum proliferation of callus (2.45g) was observed in NIA-2011 somaclone with medium containing 1.0 mg L -1 2, 4-D.However, the minimum value (0.33 g) was observed in same somaclone using concentration of 4.0 mg L -1 2, 4-D (Table 5 and Figure 3).These outcomes are trailed by Dibax and Bomfim [24] whose perceptions portrayed the embryogenic masses proliferation on MS culture medium with 2, 4-D, the diminishment of 2, 4-Dconcentration to (2.0 mg L -1 ) Effectively double the number of embryogenic masses in every subculture.6 and Figure 4).These information affirmed the outcomes reported in Primula ssp.Size of shoots length plant -1 (c) A significant decrease in size of shoot length was observed with an increase in growth regulator concentration.Stretched shoots (9.20 cm) were observed in BL4 somaclone with 1.0 mg L -1 2, 4-D concentration used.Maximum shoot elongation was also noted in NIA-2010 somaclone with the application of 2, 4-D (1.0, 2 .0 and 3.0 mg L -1 ).The clones were non-significant However, minimum shoot length (1.06 cm) was noted in NIA-11 somaclone on callus derived from 0.5 mg L -1 2, 4-D (Table 3 and Figure 4).Comparable results were likewise by Raza et al. [16] Gunderson et al. [23] in sugarcane.

Number of Chlorophyll mutant / bottle
Regeneration of albino and viridis plantlets displayed the presence of chlorophyll changes in-vitro plantlets.The chlorophyll mutant under the influence of growth regulator concentrations showed maximum numbers of chlorophyll mutants (6.40) when NIA-10 somaclone was grown on callus derived from 1.0 mg L -1 2,4-D.Minimum chlorophyll mutant was recorded in NIA-2011somaclone through application of 0.5 mg L -1 2, 4-D (1.00) (Table 8 and Figure 4).A chlorophyll lacking phenotype can be responsible for passive transformations [3, 27] additionally reported high genetic similarity among sugarcane varieties using RAPD marker.
Analysis of variance of number of root plant -1 regenerated from various concentrations of IBA with different varieties.[7] Shoed or depicted that utilization of IBA with 6% sucrose in growth medium incited fiery root improvement.The plantlets with all around created shoots and roots were transferred to jiffy pots having sterilized perlite.After acclimatization the plantlets were initially transferred to the pots for hardening and later on in the field.Highest number of bands was obtained with Primer A-02, while the lowest numbers were obtained with primers B-10 and B-08 (Figures 1 and 2).Some specific bands were also identified, thus, reflecting the RAPDs application for the identification of sugarcane mutants.Results revealed that somaclone 2010 and NIA-2011 somaclone contains a specific band of 451bp amplified with primer A-2.In the relationship between sugarcane somaclones and parent, the highest similarity was obtained between BL4P and BL4SC (96%), while minimum similarity was found between NIA-2011 and NIA -2010SC (69%).Molecular markers have turned out to be efficient tools in the characterization and estimation of genetic diversity within the species and population [18] RAPD markers have turned out to be a solid marker framework for genetic fingerprinting and furthermore, for identifying genetic diversity [19,28] reported that some RAPD bands were acquired in sugarcane families, showing that RAPD bands could effectively be utilized as genetic markers.While RAPD-PCR fingerprints have been generally utilized in diverse studies, the utility of RAPD-based bands as markers is confounded as they may not be locusspecific [29]  Together, these observations recommended either that a few genotypes are more susceptible to somaclonal variation, or that the in vitro unsteadiness is really a result of a genotype versus culture medium interaction.

Table 11. Effect of different concentration of indolebutric acid (IBA) + sugar on root induction Conclusion
Callus culture of three somaclones NIA-2011, NIA-2010 and BL4 produced genetic variability in the clone.During the research study, dry nodular and compact callus were found in two clones BL4 and NIA-2010 somaclones,while the smooth compact callus recognized type A and B callus in NIA-2011.Maximum callus and plant regeneration were observed with used 1.0 and 2.0 mg L -1. 2, 4-D.However, maximum proliferation of callus was also observed in same concentration of 2, 4-D, while increased concentration of IBA + sugar decreased primary and secondary roots.The NIA-2010, somaclone produced vigorous plants during the hardening process in greenhouse as well as, has outstanding performance in field during acclimatization process.