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Current Trends in Biotechnology and PharmacyVol. 5 (1) 1004-1010 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1007Table 3. Effect of glutamine on organogenetic response of node derived callus of guayule.MS medium with Glutamine Sucrose Number of calli Number of shootshormones (mg/l) (mg/l) (g/L) cultured formed per callusmass (250+20 mg)0.1NAA + 2.5 BAP 200 20 20 13-150.1 NAA + 2.5 BAP 200 20 25 22-250.1 NAA + 2.5 BAP 200 20 23 22-250.1 NAA + 2.5 BAP 200 20 20 10-150.02 NAA + 2.5 BAP 200 20 18 18-20*Data collected from 10 cultures per treatment.MS medium was supplemented with 0.1 mg/lNAA + 2.5 mg/l BAP + 200 mg/l glutamine and2% sucrose. NAA in combination with BAP wasfound more effective in shoot formation than BAPalone.Effect of age of callus on the percent frequencyof shoot regeneration and rooting of shoots :Shoot regeneration ability decreased from 84 to30% with an increase in the age of the callusfrom 100 to 250 days (Table 4). Shoots obtainedfrom the callus tissues were transferred to MSTable 4. Regenerating ability of node derivedcallus cultures of guayule (Partheniumargentatum Gray) from long-term cultures.Age of callus(in days)% frequency ofshoot regeneration50 8860 85100 84200 85250 30*Data collected from 10 cultures per treatment.agar medium fortified with either IAA or NAAor 2,4-D. Increasing the concentrations of IAA(0.04 mg/l to 0.5 mg/l) or NAA (0.08 mg/l to 0.5mg/l) enhanced the percentage of cultures rootingat the cut ends of shoots when incubated in dark.On the other hand, 2,4-D produced roots both inlight as well as in dark. The synthetic auxin 2,4-Din the medium (0.04 mg/l and 0.06 mg/l) seemedto be better when maintained in light comparedto higher concentrations for rooting of shoots. Thenumber of roots differentiated per shoot for threehormones is shown in Table 5. Plantlets were latertransferred to pots containing sand and soilmixture in the ratio of 1: 1. Plants were coveredwith glass beakers to maintain humidity andwatered with Hoagland nutrient solution at 3-4day intervals. Glass beakers were removed aftertwo weeks of transfer to the pots. The frequencyof survival was 60% and the plants did not showany morphological variations.Arreguin et al (21) establishedParthenium cultures from stems. Zavala et al(18) obtained axenic cultures from seedlingexplants or its cotyledons or leaf blades, petiolesand roots. MS medium supplemented with 0.5Maruthi Rao et al
Current Trends in Biotechnology and PharmacyVol. 5 (1) 1004-1010 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1008Table 5. Treatment of excised shoots with IAA, NAA, 2,4-D and the percentage ofcultures rooting at cut end.Treatment (mg/l) % of cultures rooting at cut ends Number of rootsLight Dark per culture0.02 IAA - - -0.04 IAA - 50 1.00.06 IAA - 15 1.00.08 IAA - 65 2.50.1 IAA - 70 2.50.5 IAA - 75 3.00.02 NAA - - -0.04 NAA - - -0.06 NAA - - -0.08 NAA - 70 3.50.1 NAA - 72 5.50.5 NAA - 74 6.50.02 2,4 – D 35 38 3.50.04 2,4 – D 70 45 4.50.06 2,4 – D 72 50 8.50.08 2,4 – D 60 50 9.00.1 2,4 – D 55 42 8.00.5 2,4 – D 48 30 4.0*Data collected from 10 cultures per treatment.mg/l 2,4-D and 1 mg/l BAP formed optimumcallus and growth in Parthenium hysterophorus(22). On the other hand, MS medium containing0.18 mg/l IAA and 0.02 mg/l KN showed goodgrowth of the callus (17). Callus cultures ofguayule exhibited superior growth with acombination of 1 mg/l kinetin, 0.2 mg/l NAA and2 mg/l inositol (18). In the present study, lowerconcentrations of KN, BAP (0.5 mg/l), 2,4-D (1mg/l) and NAA (0.5 mg/l) seemed good for callusinitiation and also when 2,4-D plus BAP wereused together. Subramanian et al (17) reportedshoots from stem derived callus tissues ofP.hysterophorus on MS medium incorporatedwith IAA (1.75 mg/l) plus either KN (1.01 mg/l)or BAP (1.13 mg/l). Zavala et al (18) reportedshoot formation from root or hypocotyl derivedcallus tissues of P.argentatum on Mahlberg’smedium fortified with KN (0.1 mg/l), 2,4-D (0.2mg/l), NAA (0.2 mg/l) and 2 mg/l inositol. MSmedium containing 0.1 mg/l KN and 10 mg/l 2(3,4-dichlorophenoxy) triethylamine derivative obtainedshoots from callus (23). Shoot differentiation onMS medium supplemented with 0.1 mg/l BAPwas reported by Staba and Nygaard (24). In thepresent study, addition of glutamine (200 mg/l)resulted in the highest frequency (84-88%) ofshoot regeneration. There is an increase in theRegeneration in Parthenium argentatum
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