EFFECTS OF DIFFERENT PHYTOHORMONES ON SUGARCANE (SACCHARUM SPP.) REGENERATION

Three sugarcane clones, AEC82-223, Ghulabi-95 and AEC86-328 were investigated for callus induction and regeneration under different concentrations (2mg/L and 4mg/L) of various auxins i.e. 2,4-dichlorophenoxyacetic acid (2,4-D), indol acetic acid (IAA), 3,6dichloro 2-methoxy benzoic acid (Dicamba) and picloram. An efficient callus induction was observed on the medium containing picloram followed by 2,4-D but high callus proliferation was achieved with picloram. Non-regenerable callus was produced when calli were transferred for proliferation on Dicamba. The maximum calli induction, its proliferation and plantlets regeneration were recorded in clone AEC82-223, while the minimum in AEC86328. The maximum chlorophyll mutation frequency was noted in clone AEC86-328 and minimum in AEC82-223. Among auxin especially 2,4-D induces more genetic variability as compared to other auxins used in this work. INTRODUCTION Sugarcane (Saccharum spp. hybrid) is one of the most important cash crops of Pakistan. Average cane yield and sugar recovery in Pakistan are the lowest among the sugarcane growing countries of the world (Anonymous, 2002). Because of nonor sporadic flowering, natural viable fertile seed production has ever been a problem in Pakistan. Hence, alternative methods such as in vitro culture techniques and induced mutations are being employed to create the new genetic variability for the selection of desired clones/genotypes of sugarcane (Khatri et al 2002). Realization of the full potential of somatic cell genetics in higher plants is predicted on the ability to induce desired development state (Orton, 1979). Callus has now been induced in a large number of sugarcane species indicating that, this phenomenon is not limiting (Narayanvaswamy, 1977). The fascinating feature of callus culture is that one can alter one or few character (s) of the questioned genotype, keeping the rest of the genome intact. Ahloowalia (1995) reported that the development of desired genotypes is only possible through somaclonal variation or through in-vitro mutagenesis in case of vegetatively propagated sugarcane plants. The ability to regenerate the plantlets from callus tissue of Saccharum species was first demonstrated by Heinz and Mee (1969). Liu and Chen (1976, 1978, 1984) have reported significant variations in somaclones in the important agronomic characters such as cane yield and its components, sugar contents and some morphological traits. The objective of the present work was to assess the effects of different auxins on callus induction and plant Sugarcane Tissue Culture Pak. J. of Biotechnology 18 differentiation in different genotypes of Saccharum spp (hybrid) under tissue culture conditions. MATERIALS AND METHODS Three sugarcane clones, AEC82223, AEC86-328 and Ghulabi-95 were used for tissue culture studies. Ten explants containing leaf primordia were taken from each genotype, sterilized by standard procedure (Siddiqui et al., 1988) and cultured on modified MS medium (Murashige and Skoog, 1962) containing 2 mg/L and 4mg/L, 2,4-D, dicamba, picloram and indole acetic acid for callusing. Media was solidified with 0.8% Difco bacto agar. Commercial sugar was used instead of Analar grade sucrose as carbon source in the medium. After five weeks of explantation, the calli were weighed and cultured on shoot induction medium (MS +2 mg/L IBA + 2 mg/L IAA + 2 mg/L kinetin). The regenerated shoots were scored for chlorophyll mutations. When the plantlets attained 7-8 cm height, these were subjected to rooting by culturing on different media viz.; i) MS medium, ii) 1⁄2 MS medium, iii) MS medium + 1mg/L IBA + 3% sugar, iv) MS +1mg/L IBA+ 4% sugar, v) MS +1mg/L IBA + 5% sugar, vi) MS + 1 mg/L IBA + 6% sugar, vii) MS + 1 mg/L IBA + 7% sugar and viii) MS + 1 mg/L IBA + 8% sugar. All these operations were carried out under aseptic conditions and cultures were incubated at 28 ± 2C with 16 hours photoperiod. Rooted plantlets were acclimatized and transplanted to field. RESULTS AND DISCUSSION Callus induction: Based on morphological appearance, two types of calli were observed: (i) type A-yellowish white, compact, dry and nodular (Fig 1) and (ii) type Bwhitish globular, noncompact and wet(Fig. 2). Such type of calli have also been reported by Khan et al., 1998 and Khatri et al., 2002. Best callus induction and proliferation was observed on medium containing 4mg/L 2,4-D and 4mg/L picloram. Siddiqui et al., 1988 and Begum et al., 1996, have reported similar results. Picloram in 2mg/L also yielded good callus. AEC82223 yielded the maximum callus followed by Ghulabi-95 while AEC86-328 produced the minimum (Table 1). No callus induction was observed in indole acetic acid medium. Dicamba yielded very few calli in AEC82-223 and Ghulabi-95 but on second transfer the calli were turned into non-regenerable callus. Similar trend was observed in callus proliferation on sub-culture. Callus weight reduced in Ghulabi-95, because of high percentage of type B callus. According to Orton (1979), the type B callus of Hordeum vulgare has twice intrinsic growth rate as compared to type A, but in our study, it was observed that type B callus of sugarcane did not exhibit the same attributes, rather its growth substantially decreased with similar results vclones AEC86-328 yielded type A callus on dicamba, but on subculture it got converted into type B. Aging of the medium affected morphological status of callus in AEC86-328, whereas calli of AEC82-223 and Ghulabi-95 were converted into somatic embryos.