439

ISSN: 0974 - 0376

KEYWORDS

Proceedings of International Conference on

Anthropogenic Impact on Environment & Conservation Strategy

(ICAIECS - 2012)

November 02 - 04, 2012, Ranchi,

organized by

Department of Zoology, Ranchi University, Ranchi

&

Departments of Zoology and Botany,

St. Xavier’s College, Ranchi

in association with

National Environmentalists Association, Indiawww.neaindia.org

NSave Nature to Survive

: Special issue, Vol. 1:

www.theecoscan.inAN INTERNATIONAL QUARTERLY JOURNAL OF ENVIRONMENTAL SCIENCES

Ankita Kumari et al.

Glycine max

Soyabean

Callus

MS Medium

Auxins

439 - 443: 2012

CALLUS INDUCTION AND REGENERATION POTENTIALITY OF

GLYCINE MAX (L) MERR.-A PLANT WITH HIGH NUTRITIVE VALUE

440

ANKITA KUMARI, ICHHA PURAK, ARUNA SINGH AND SUPRIYA SHRIVASTAVA*

Centre for Biotechnology, Ranchi Women’s College, Ranchi - 834 001E-mail: bhasha_sh@yahoo.co.in

*Corresponding author

NSave Nature to Survive QUARTERLY

Glycine max (L) Merr (Soyabean) of family

Fabaceae is a significant legume crop exten-

sively cultivated for food, forage and soil im-

provement but especially for its nutritious pro-

tein and oil-rich seeds. Soybean is a rich source

of dietary proteins including all essential amino

acids. It contain isoflavones such as genistein

and daidzein having estrogen like effects. Soy-

bean oil is recommended for cooking pur-

pose as it contains polyunsaturated alpha li-

nolenic acid which reduces cardiovascular

risk. In the present investigation attempts have

been made to standardize a protocol for pro-

duction of callus mass and regeneration of

Glycine max. Effects of different auxins (2, 4-

D, NAA, IAA and IBA) on its various explants

viz. root, stem and leaves have been analyzed.

Auxin concentrations ranging from 0.5 mg/L

to 5mg/L were employed. 2, 4-D has proved

itself superior to NAA, IAA and IBA in pro-

ducing callus. Stem and leaves are suitable

explants for regeneration.MS media supple-

mented with 2.5 mg/L IAA taking stem as ex-

plant was able to produce callus which turned

greenish in colour and ultimately whole plant

was developed with shoot, root and leaves

within six weeks of culture

ABSTRACT

INTRODUCTION

Glycine max (L) Merr. (Soyabean) is erect bushy hairy annual herbaceous grainlegume having trifoliate leaves and purple to pink flowers; extensively cultivatedfor food, forage and soil fertility improvement but especially for its nutritious oil-rich seeds. It is native to East Asia. Main producers of Glycine max are USA (35%),Argentina (27%), Brazil (19%), China (6%) and India (4%). Its seeds are used assource for biodiesel production. It is rich source of dietary proteins. It is used asraw as well as processed food and also in drinks. Traditional non fermented usesof soybean as food source products include soya sauce, fermented bean paste,tempeh and natto. USA is largest consumer of soya food products. Warm climate issuitable for its cultivation but water logging inhibits its growth and is also suscep-tible to draught, high and low temperature, insect pests, weeds and various patho-gens. Soyabean contains protein, isoflavones, include soya milk, soya flour andtofu etc. Non traditional fermented food and fiber, all thought to provide healthbenefits. It is an excellent source of dietary protein, including all essential aminoacids. It is also a source of phospholipid lecithin. Isoflavones such as genistein anddaidzein are believed to have estrogen like effects in the body so are sometimescalled as phytoestrogens.These plant estrogens though having mild effect are ad-vised by some dieticians to be usefull to prevent cancer and solve post menopauseproblems (Duke,1981). In order to enhance the production and also improve thequality and quantity of useful ingredients and to overcome biotic and abiotic chal-lenges a systematic and sustainable crop improvement plan is to be followed.Soyabean is conventionally propagated through seeds. Improvement of cropsthrough plant tissue culture can be accomplished by growing plants in the labora-tory through callus initiation and regeneration of shoots and roots or regenerationof multiple plants directly from explants such as leaves, shoots, seeds, embryosand cotyledons. As it is a legume having seeds rich in protein and oil content so isa valuable farm product in high demand. To meet the increasing demand needs tobe cultured in- vitro so as enormous plantlets of superior varieties having resis-tance can be supplied to farmers. Kudirka et al. (1986) have studied interaction ofAgrobacterium tumefaciens with Soybean leaf explant culture for genetic improve-ment. Olhoft et al. (2006) have tried for agrobacterium mediated genetic transfor-mation of Soybean using mature cotyledonary node explants. As Glycine max

(Soybean) is a plant with increasing demands due to its multipurpose uses, there isurgent need for alternative system of multiplication of this plant. Keeping this inmind, in the present investigation attempts have been made with an objective tostandardize a protocol for production of callus mass and regeneration of soyabean.There are a few earlier reports on in- vitro regeneration of Glycine max (Barwale et

al., 1986; Wright et al. 1987; Dan and Reichert ,1998; Kim et al., 2004; Kumari et

al., 2006; Kita et al., 2007; Radhakrishnan and Ranjithakumari, 2007; Loganathanet al., 2010). In the present project MS solid media supplemented with differentcombinations of auxins viz. 2, 4-D, NAA, IAA and IBA have been tried usingvarious explants such as root, stem and leaves.

441

CALLUS INDUCTION AND REGENERATION POTENTIALITY OF GLYCINE

Callus scoring += 1-20%, ++= 21-50%, +++= 51-80%, ++++= 81-100%

Table 1: Callus response of Glycine max explants in MS Media supplemented with different concentration of Auxins after 4-6 weeks of culture

Auxin Concentration mg/L Callus scoring Morphogenic response of callus

2,4-D

0.5 +

1.0 ++

1.5 ++++ Compact callus

2.5 ++++

5.0 ++++ Cream coloured callus turned brown when mature

NAA

0.5 +

1.0 +

1.5 ++

2.5 ++ Callus initially of cream colour. green patches appeared later

5.0 ++ Shoots developed from callus mass

IAA

0.5 +

1.0 +

1.5 ++

2.5 ++ shoots, roots and leaves regenerated from callus

5.0 ++

IBA

0.5 +

1.0 + creamish white friable callus developed less in amount

1.5 ++

2.5 ++

5.0 ++

MATERIALS AND METHODS

Culture medium

MS (Murashige and Skoog, 1962) basal medium was used inthe present investigation. Concentrated stock solution of majorand minor salts, iron source, vitamins and amino acids wereprepared separately and stored under refrigeration. Requiredvolume of stock solution was pipetted out during mediapreparation and 0.3% sucrose was added. The chemicalsused for preparing media were of analytical grade from Loba,Merck and Sigma. Medium was homogenized by boiling andcontinuous stirring with pH of medium adjusted upto 5.8before adding 0.8% agar by using 0.1 N NaOH or 0.1N HCL.Desired concentration of growth regulators was added andmixed properly. 15-20mL medium was poured into culturetubes (15x2.5cm) which were washed thoroughly and rinsedin distilled water and oven dried. Sterilizarion of medium wasdone for 15- 20 minutes at pressure 1.1 kg/cm2 at temperature121ºC. Tubes containing sterilized medium were left in tiltedposition to prepare slants in air conditioned room.

Preparation of Explant

Some seeds of Glycine max (L) Merr. collected from seedcentre, Ranchi were soaked in water to facilitate in-vitrogermination. Vegetative parts such as stem, leaf and root weretaken as explant from in vitro grown seedlings. The explantsafter thoroughly washing with tap water were treated withSuthol solution for about 5 minutes and again washed withrunning tap water. The explants were then surface sterilizedin 0.2% of HgCl

2 by immersing for 2-3 minutes and then were

rinsed in double distilled water. These sterilized stem andleaves were then cut into pieces of about 1.0-1.5cm and madeready for inoculation in order to observe callus formationand regeneration potentiality. Various concentrations rangingfrom 0.5mg/L to 5mg/L of four auxins (2,4-D, NAA, IAA andIBA) were used. Seedlings used for obtaining explants were

grown in culture tubes aseptically as well as in pots. Seedswere soaked in water for 24h before inoculation. Fullydeveloped seedlings formation took about 15 days. Root, Stemand leaf explants were used for callus induction andorganogenesis

Inoculation

The explants were in-oculated into test tubes containingsterilized media under Laminar Air Flow cabinet. Transfer areawas sterilized with UV light and by swabbing the floor surfacewith 95% ethyl alcohol. Inoculation tools were flamed beforetransferring the explants. Each experiment with 10 culture tubeswas repeated 4 times and were observed every day for their

morphological response.

RESULTS

In the present investigation 2, 4-D appeared as most suitablefor callus induction followed by NAA, IAA and IBA. 2.5 mg/Lto 5 mg/L 2, 4-D was found to be optimum for callus growthtaking root as explants (Fig.1). Few small secondary roots alsoappeared in about 4-6 weeks cultures. 2, 4-D induced callusformation in both leaf and stem explants (Fig. 2 and 3). 2, 4-Dand NAA both initiated callus either alone or in combination.Callus produced was of cream colour in primary stage whichgradually turned brown or yellowish green (Table-1). NAA,IAA and 2, 4-D helped in root initiation whereas IBA was foundto be suitable for formation of shoots. NAA was able to induceconsiderably good amount of callus in stem explants. 5 mg/LNAA was also able to induce leaves within 6 weeks (Fig. 4).The callus was of cream colour initially. After 4 weeks fewgreen patches appeared at some parts of callus. From this sub-cultured callus roots about 1" in length developed. In case ofleaf explant comparatively less amount of callus was producedbut roots were not developed. In case of root explants callusgrowth was very poor. Regeneration potential of IBA was more

442

ANKITA KUMARI et al.,

and resulted in formation of roots and shoots both. Callusproduction was less, cut ends of leaves developed only callus,a few white coloured roots also developed. Stem and leavesboth regenerated on MS medium supplemented with IAA andattained maturity. Stem explants produced both roots andshoots in concentrations ranging from 1.5 mg/L to 5mg/L. IBA(Figs. 5 to 8).

DISCUSSION

Callus induction by 2, 4-D and NAA concentrations in the

present study have been earlier reported by Ebony et al. (2010)who have taken excised cotyledons and embryos from

germinating seeds and have reported formation of callus withvarious concentrations of 2, 4-D and NAA used alone or incombination. The callus was further transferred to BAP andkinetin containing media to obtain roots and shoots. Finallyfully developed plants were transferred to pots in less than 3months. In present investigation MS media supplemented with2.5 mg/L IAA taking stem as explant was able to producecallus (Fig. 9) which turned greenish in colour and ultimatelywhole plant was developed with shoot, root and leaves withinsix weeks of culture (Figs. 10 to12). Isler and Vural (2010)were able to obtain plantlets of Glycine max by germinationgseeds in-vitro condition on MS hormone free medium within21 days .Part of these sterile plantlets were used by them as

Figure 1 to 12: 1. Callus developed from root explants on M S medium containing 2.5mg/L 2, 4-D 2. Callus developed from stem explant on

MS medium containing 5mg/ L 2, 4-D in 4-6 weeks 3. Callus developed at the cut ends of leaf explants by medium containing 5mg/L 2, 4-D

4.Formation of callus and induction of leaf within 6 weeks taking stem as explants by 5 mg/L NAA 5. Induction of primary and secondary

root,leaves and little callus by 2.5 mg/L IBA after 7 days when stem taken as explants 6. Callus and leaves induced by 2.5 mg/L IBA after 21

days with stem taken as explants 7.Induction of roots and leaves by 2.5 mg/L IBA after 42 days 8. Induction of callus within 4 weeks using leaf

explants and 5mg/L IBA 9. Formation of callus and leaves by 1.5 mg/L IAA when stem taken as explants 10 Effect of 2.5 mg/L IAA on stem

explant showing roots and leaf development in 4 weeks 11. Development of whole plant within 4-6 weeks by leaf explants 12. Development

of whole plant with numerous leaves, well developed roots coming out from callus by2.5 mg/L IAA within 4 weeks

1 2 3 4

5 6 7 8

10 11 129

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CALLUS INDUCTION AND REGENERATION POTENTIALITY OF GLYCINE

explants in tissue culture experiments with various auxincombinations.The results of present project reveal MS mediumsupplemented with auxins IBA or IAA can be safely employed

for earlier regeneration purposes

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