Effect of Different Auxin Concentration on Rooting in Pigeonpea ( Cajanus cajan [ L . ] Millsp . ) cv . Manak ( H 77216 ) Via Cotyledonary Node Explants

Pigeonpea (Cajanus cajan [L] Millsp.) is an important grain legume of the semi-arid tropics. It provides protein rich food. Seeds of pigeonpea were collected and surface sterilized, these sterilized seeds were germinated aseptically in shoot induction media (SIM) fortified with MS media supplemented with 2mg/l BAP. Cotyledonary node explants (excised from 15 day old seedlings) were transferred to shoot elongation media (SEM) for elongation fortified with MS media + 1.0 mg/l GA3. Elongated shoots were transferred to root induction media (RIM) using different concentration of IAA and IBA. Highest percentage (93) of rooting was observed on modified MS media + 1.0 mg/l IBA.


INTRODUCTION
Pigeonpea [Cajanus cajan (L.) Millsp.] with chromosome number of 2n=22, is an important grain legume crop of rainfed agriculture in the semi-arid tropics of the Indian sub-continent and is also widely grown in eastern and southern Africa, Latin America and the Caribbean (Nene and Sheila, 1990).Pigeonpea is cultivated in more than 25 tropical and subtropical countries, either as a sole crop or intermixed with cereals, such as sorghum (Sorghum bicolor), pearl millet (Pennisetium glaucum), or maize (Zea mays), or with other legumes, such as peanuts (Arachis hypogaea).Being a legume, the pigeonpea enriches soil through symbiotic nitrogen fixation.
Ripe seeds are eaten fried or boiled, often after being soaked first, or boiled into porridge.In the Indian subcontinent pigeonpea is mainly used as a pulse, in the form of 'dhal' or 'dal' (soaked, dried, hulled and split seeds), and this use is carried on by Indian communities in Africa.Pigeonpea is useful in hedges and windbreaks on dry soils and in agroforestry (e.g. in alley cropping systems, where it is pruned to supply green manure).It is also grown as a shade crop, cover crop, or as support for vanilla.
Shoots developed in presence of higher cytokinin to auxin ratio generally lack roots that prevent the generation of complete plantlets.In general, the rooting medium has low salt e.g.½ or even ¼ salts of the MS medium, and reduced sugar levels (usually 1.0 g/L).Generally 0.1-1.0mg/L NAA or IBA is required for rooting.Shoots are usually rooted in an agar medium, but the recent trend is to root them directly in vermiculite or potting mix.The cut ends of shoots are treated with a suitable auxin solution or powder mix, transplanted in pots and kept under high relative humidity and low light intensity.This saves cost as rooting and soil transfer stages are combined and rooting medium is eliminated.
In 1957, Skoog and Miller described the essentiality of plant growth regulators concentration in culture media.The relative concentration is very critical for growth and morphogenesis.The ratio of cytokinin to auxin depicts the occurrence of changes in plants.The higher cytokinin to auxin ratio is found to be suitable for shoot regeneration.Usually, the following growth regulators were used in pigeonpea regeneration: auxins like IAA (Mohan and Krishnamurthy, 1998;Yadav and Padmaja, 2003;Dayal et al., 2003), IBA (Shivprakash et al., 1994;Geetha et al., 1998), NAA (George and Eapen, 1994) and 2,4-D (Anbazhagan and Ganapathi, 1999) were used in various combinations with cytokinins like kinetin (Mohan and Krishnamurthy, 1998;Geetha et al., 1998;Dayal et al., 2003;Villers et al., 2008), BAP (Shivaprakash et al., 1994;Geetha et al., 1998;Pudukottai, 1998;Mohan and Krishnamurthy, 1998), TDZ (Eapen et al., 1998) to promote cell division, regeneration of shoots and to enhance proliferation and growth of auxiliary buds.
Hence, the present study aims to attempt produce of cotyledonary node explants of pigeonpea cv.Manak (H77216) from suitable auxin combination for in vitro regeneration study.

Plant materials and culture conditions
Seeds of pigeonpea variety Manak (H77216) were used for all the experiments in the present studies.Unless mentioned otherwise, all media contained MS salts and organic constituents (Murashige and Skoog, 1962), 3% sucrose, 0.7% (w/v) agar, and the pH was adjusted to 5.8 before autoclaving.For explant culture and shoot bud development, 50ml conical flask were used for seed inoculation closed with plugs made with non-absorbent cotton.For shoot elongation and rooting, 250ml conical flask were used closed with plugs made with nonabsorbent cotton.All the growth regulators including BAP, GA 3, IAA and IBA were added after filter sterilization with nylon filter (Millipore) of 0.22µm pore size.The cultures were incubated at 25±2°C temperature, with a light regime of 45-60µE/m 2 /s for 16 h and 8 h at dark.

Seed sterilization
Seeds of pigeonpea were collected and washed thoroughly under running tap water for 10 min and washed with autoclaved double distilled water 2-3 times.Then rinse in 1-2 drops of Tween-20 (liquid detergent) for 20 min and wash with tap and distilled water until all foam is removed.Then wash seeds with 70% alcohol for 5 min, followed by treatment with a solution of 0.1% (w/v) NaOCl (bleach) for 5 min and finally with autoclaved distilled water in laminar air flow.The seeds were soaked for overnight in autoclaved distilled water.

Explant preparation and shoot regeneration
Seed coats from the pre-soaked seeds were removed under aseptic conditions and seeds were inoculated in media comprising of MS, B 5 vitamins, 2mg/l BAP, 3% (w/v) sucrose and 0.7% agar.Embryonic axes obtained from overnight soaked sterilized seeds.Embryonic axes were further excised by removing radicle apex and plumule apex along the axis resulting in two explants per seed.

Elongation of shoots
In the present study, BAP alone was found to be suitable for both multiple shoot bud induction and proliferation.However, the multiple shoots obtained on various concentrations of BAP failed to elongate on the same medium resulting in rosettes of shoots.Hence it was necessary to develop suitable media for proliferation and elongation of shoot buds.Clumps of multiple shoots were transferred to conical flask containing 1mg/l GA 3 for elongation.

Rooting of shoots
Elongated and well-developed shoots (> 3 cm long) were excised from the shoot clumps and transferred to MS medium augmented with various concentrations of IAA and IBA alone for root initiation.The hormone combinations used were [1mg/l, 2mg/l & 3mg/l] & IBA [1mg/l, 2mg/l & 3mg/l] IAA in RIM1, RIM2, RIM3, RIM4, RIM5 and RIM6 media respectively.The rooting frequency was further calculated.(Table I).

RESULTS AND DISCUSSION
Seeds inoculated in SIM media containing MS, B 5 vitamins, 2mg/l BAP, 3% (w/v) sucrose and 0.7% agar produced explants after 15 days.Each explant was then transferred to SIM for 7 days and then to SEM fortified with 1.0 mg/l GA 3 for 15 days.Each elongated shoot was then transferred to rooting media with different auxin at different concentration.Elongated shoots transferred to RIM1 media fortified with 1mg/l IBA showed maximum rooting.RIM1 media gave thickest and the most developed rooting.

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Likewise, elongated shoots transferred to RIM4 media fortified with 1mg/l IAA showed no rooting.Similarly, elongated shoots transferred to RIM5 media fortified with 2mg/l IAA and RIM6 media fortified with 3mg/l IAA showed no rooting as shown in Fig. 4, 5, 6.
It is observed that elongated shoots did not respond to IAA while shoots only showed rooting in media containing IBA.On lower concentration of IBA i.e., 1mg/l 93.0 % rooting was obtained whereas 2mg/l IBA produced minimum of 53.0 % of rooting was obtained.Similarly, at 3mg/l, 73.0 % rooting was obtained as shown in Fig. 7.
It was observed that Manak variety showed indeterminate results i.e., in 1mg/l IBA highest rooting frequency is obtained while at 2mg/l IBA lowest rooting frequency is obtained and at 3mg/l IBA medium rooting frequency is obtained.So, we can definitely assign Manak as indeterminate variety.

TABLE I :
Influence of auxin on rooting of in vitro derived shoots of pigeonpea after 2 weeks of culture While elongated shoots transferred to RIM2 media fortified with 2mg/l IBA gave minimum rooting.Roots in RIM2 media were very weak and underdeveloped.But elongated shoots transferred to RIM3 media fortified with 3mg/l IBA gave comparatively more thick rooting but less dense as in RIM1 media as shown inFig 1, 2, 3.