full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 5 (1) 1004-1010 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1005stresses. Efficient methods of plant regenerationfrom tissue cultures are necessary for theselection or the creation of useful variants andalso for taking up genetic engineering studies.Subramanian and Zavala et al (17, 18) reportedParthenium callus cultures that developed rootsand shoots. Mass propagation of guayule by invitro organ and callus cultures was developed byDhar et al (19). Present study deals with theeffect of different growth regulators, glutamineand sucrose concentration on the regeneration ofshoots from node derived callus cultures ofguayule.Materials and MethodsNodal explants measuring 0.5 to 0.8 cm from2-year-old shrubs of guayule were surfacesterilized with 0.1% mercuric chloride for 6-8minutes followed by washing with sterile glassdistilled water. The explants were inoculated oneinto each test tube containing 15 ml of MS agarmedium (20) supplemented with differentcombinations and concentrations of growthregulators. The pH of the medium was adjustedto 5.8 before solidifying with 0.8% agar. Callustissues initiated from the nodal explants were subculturedroutinely onto MS medium containing 0.2mg/l NAA + 0.2 mg/l IAA + 1 mg/l BAP andincubated at 26 ± 2 0 C. Callus was sub-culturedfor every 25-30 days. For generation of shoots,approximately 250 ± 20 mg of callus was used.For rooting of the in vitro derived shoots, half theMS salt concentration but with full strength ofiron was used with varying concentrations ofauxins either in light (30 µE m -2 s -1 ) or in dark at26 ± 2 0 C. Before transferring the rooted shoots,they were washed thoroughly to remove theadhered agar if any into earthen pots containinga mixture of soil, sand and farmyard manure inthe ratio of 1:1:1 and grown in the net-house foracclimatization. Hoagland solution was addedduring this period once in every 2-days.Percentage survival of plants was recorded afterone month of transfer to the pots.Results and DiscussionEffect of growth regulators on callus initiationfrom nodal explants : NAA or 2,4-D alone, 2,4-D and BAP together produced only callus but notorganogenesis. With an increase in theconcentration of KN and BAP from 1 mg/l to 5mg/l, there is a gradual decrease in the frequencyof callus production (Table 1) but increase in themultiple shoot induction directly from the nodalexplants (data not shown). Nodal explantsproducing only callus with different plant growthregulators is 75% with 0.5 mg/l KN; 90% with0.5 mg/l BAP; 70% with 1 mg/l 2,4-D; 50% with0.5 mg/l NAA + 0.5 mg/l BAP but 92% with 0.5mg/l NAA.Effect of sucrose and glutamine on number ofshoots formed per callus mas : The number ofshoots formed per callus mass (250 + 20 mg) isshown in Table 2. BAP at the concentrations of0.1 mg/l and 1 mg/l in the medium (devoid ofglutamine) exhibited 49% and 58% frequency ofshoot regeneration respectively. The frequencyof shoot regeneration from the callus of guayuleincreased considerably when 0.1 mg/l NAA and2.5 mg/l BAP were used together. Sucrose at0.5% did not show any organogenetic response.Shoot differentiation was 85% with theincorporation of 0.1 mg/l NAA + 2.5 mg/l BAP(devoid of glutamine) along with 4% sucrose inthe medium. However, 6% sucrose drasticallyreduced the shoot forming ability of the callus.Addition of 200 mg/l of glutamine greatly improvedthe number of shoots from 12-15 to 22-25 percalli (Table 3). Thus, the concentration ofglutamine and sucrose in the medium greatlyaffected the percentage frequency of shootregeneration in guayule. The frequency of callishowing shoot regeneration is high (88%) whenMaruthi Rao et al