THIDIAZURON-INDUCED HIGH FREQUENCY SHOOT REGENERATION FROM LEAF AND NODE DERIVED CALLUS OF PUERARIA TUBEROSA DC, WILLD, A MEDICINALLY IMPORTANT WOODY

Dr. V. Sadguna 1 and * Dr. Md. Mustafa 2 . 1. Doctor of Philosophy in Botany, Department of Botany, Kakatiya University, Warangal, 506009, Telangana State, India. 2. Assistant Professor in Botany, Department of Botany, Kakatiya University, Warangal, 506009, Telangana State, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

744 different concentrations and combinations of auxins and cytokinins and casein hydrolysate. The P H of the medium was adjusted to 5.8 before the addition of agar agar and culture medium was autoclaved at 121⁰C with 15 Lbs pressure for 20 minutes. After inoculation all culture tubes were incubated at 25±2⁰C, 16 hrs light/8 hrs dark of photoperiod with illumination of 1500-3500 lux fluorescent tube lights.

Resuls:-
Callus induction: leaf and node explants of Pueraria tuberosa were cultured on MS medium fortified with 2, 4-D, NAA and IAA alone and incombination with BAP. Different types of calli were produced from leaf and node explants after two subcultures each with four weeks. Green compact callus was induced on MS medium fortified with 2.5mg/l IAA + 2.0mg/l BAP and 2.0mg/l NAA + 2.0mg/l BAP. This callus was utilized for further regeneration studies.
In Vitro Shoot Regeneration:-Effect of BAP, Kn, TDZ incombination with IAA:-Green compact callus of leaf and node derived callus of P. tuberosa was cultured on MS medium supplemented with 2.5 mg/l BAP + 1.0 mg/l IAA for the induction of shoots. Few small sized shoot buds were developed after four weeks of culture from leaf and node derived callus. These buds were developed into shoots after two weeks of culture on the same medium after one passage (Table-1 , Fig-1a). The mean number of shoots were higher from leaf derived callus than node derived callus. Higher mean number of shoots (4.42 ± 0.34) were achieved from node derived callus on MS medium fortified with 2.0 mg/l Kn + 1.0 mg/l IAA of P. tuberosa (Fig-1).
The most effective (5.12 ± 0.23) in vitro shoot bud regeneration was achieved from leaf derived callus on MS medium fortified with 2.0 mg/l TDZ +1.0 mg/l IAA after two passages. The higher and lower concentration of TDZ did not promote in the proliferation of shoot buds in Pueraria tuberosa (Table-1 , Fig-1b).

Effect of BAP, NAA, IAA incombination with Casein hydrolysate:-
When the callus was inoculated on MS medium supplemented with 1.5 mg/l BAP + 200 mg/l Casein Hydrolysate (CH), the callus proliferated into shoot buds without leaching of phenolic compounds after two subcultures (Table-2, Fig-2). By using the concentration of BAP was increased from 1.5mg/l to 2.0mg/l, the mean number of shoots were increased both from leaf and node derived callus cultures (Fig-1c). The further enhancement in the mean number of shoots was achieved on MS medium supplemented with 2.0 mg/l NAA and 200mg/l CH. MS medium fortified with IAA (2.0mg/l) with 200mg/l CH also promoted shoot buds induction in both the cases after six weeks of culture with lower frequency. MS medium fortified with (2.0 mg/l) NAA and (200mg/l) CH also promoted shoot buds induction from both leaf and node derived callus after six weeks of cultures. Among the all combinations and concentrations the most effective shoot bud regeneration was achieved in MS medium supplemented with TDZ incombination with IAA from leaf derived callus and NAA in combination with CH from node derived callus of Pueraria tuberosa.

Effect of various auxins on rooting:-
In vitro shoot cultures were used for induction of rooting. The in vitro root initiation was observed after 20-30 days of inoculation of rooting medium. We have tested different concentrations and combinations of auxins, the highest mean number of roots (4.05 ± 0.18) was observed on ½ strength MS medium fortified with 3.0 mg/l IBA in Pueraria tuberosa (Fig-1d). After rooting, plantlets were transferred for hardening and acclimatization.

Hardening and Acclimatization:-
The in vitro rooted plants with fully expanded leaves and well developed roots were removed from culture tubes, without damaging root system and rinsed with sterile distilled water to remove adhering nutrient agar-agar medium and then transferred into the polycups with compost vermin and autoclaved soil in the ratio of 1:3. The polycups were covered with polybags to maintain high humidity and kept in culture chamber. They were gradually exposed from artificial environmental conditions to natural acclimatization (Fig-1e & 1f).

Discussions:-
The higher number of (4.35 ± 0.23) was proliferated from leaf derived callus on MS medium fortified with 2.5 mg/l BAP + 1.0 mg/l IAA, after four weeks of culture. Similar results were observed on MS medium fortified with BAP in combination with IAA from petiole explant of Phaseolus (Mamun et al., 2004). Veltcheva and Svetleva, 2005, 745 were reported from cotyledon derived callus on MS with different concentration of BAP with IAA (Albizia lebbeck). Priyanka et al, were observed highest mean number of shoots from leaf derived callus cultures on MS medium fortified with different concentrations of BAP in combination with Kn (Psorolia corylifolia). We have observed the moderate shoot proliferation on MS medium fortified with TDZ with IAA in Pueraria tuberosa. Higher mean number of shoot buds (5.12 ± 0.24) were proliferated on MS medium supplemented with 2.0 mg/l TDZ + 1.0 mg/l IAA from leaf derived callus of Pueraria tuberosa. Similarly maximum mean number of shoots with isoflavonoids production was reported on MS with TDZ with IBA from Pueraria tuberosa (Udomusk et al., 2009). Mohamed et al (2006) were reported that the effective regeneration on MS medium supplemented with BAP with TDZ induced highest mean number of shoot formation in Phaseolus angularis. Tzitzikas et al (2004) were also observed to stimulate bud regeneration and shoot multiplication on MS medium supplemented with TDZ with BAP or any other growth hormones of Pisum sativum. Alone TDZ also proliferated shoot elongation from Psoralea corylifolia (Shinde et al., 2009).
In our investigation shoot buds were also proliferated from leaf and node callus of Pueraria tuberosa on MS medium supplemented with BAP with CH without leaching phenolic compound were in the medium. Vasanth et al.,