An efficient protocol for the regeneration of whole plants of chickpea (Cicer arietinum L.) by using axillary meristem explants derived from in vitro-germinated seedlings

SummaryAn efficient and reproducible protocol for the regeneration of shoots at high frequency was developed by using explants derived from the axillary meristems from the cotyledonary nodes of in vitro-germinated seedlings of chickpea (Cicer arietinum L.). Culture conditions for various stages of adventitious shoot regeneration including the induction, elongation, and rooting of the elongated shoots were optimized. The medium for synchronous induction of multiple shoot buds consisted of Murashige and Skoog basal medium (MS) with low concentrations of thidiazuron (TDZ), 2-isopentenyladenine (2-iP), and kinetin. Exclusion of TDZ and lowering the concentration of 2-iP and kinetin in the elongation medium resulted in faster and enhanced frequency of elongated shoots. Cultivation of the stunted shoots on MS with giberellic acid (GA3) increased the number of elongated shoots from the responding explants. pH of the medium played a very crucial role in the regeneration of multiple shoot buds from the explants derived from cotyledonary nodes. A novel rooting system was developed by placing the elongated shoot on a filter paper bridge immersed in liquid rooting medium that resulted in rooting frequency of up to 90%. A comprehensive protocol for successful transplantation of the in vitro-produced plants is reported. This method will be very useful for the genetic manipulation of chickpea for its agronomic improvement.

[1]  P. Hasegawa,et al.  The role of sucrose and nitrogen in adventitious root formation on cultured rose shoots , 2006, Plant Cell, Tissue and Organ Culture.

[2]  Sampa Das,et al.  Expression of cryIA(c) gene of Bacillus thuringiensis in transgenic chickpea plants inhibits development of pod-borer (Heliothis armigera) larvae , 1997, Transgenic Research.

[3]  K. Barna,et al.  Whole plant regeneration of Cicer arietinum from callus cultures via organogenesis , 1994, Plant Cell Reports.

[4]  P. Kirti,et al.  Plant regeneration via somatic embryogenesis in chickpea (Cicer arietinum L.) , 1994, Plant Cell Reports.

[5]  A. Sagare,et al.  Plant regeneration via somatic embryogenesis in chick pea (Cicer arietinum L.) , 1993, Plant Cell Reports.

[6]  K. Barna,et al.  Somatic embryogenesis and plant regeneration from callus cultures of chickpea (Cicer arietinum L.) , 1993, Plant Cell Reports.

[7]  G. Frugis,et al.  Genetic transformation in the grain legume Cicer arietinum L. (chickpea) , 1993, Plant Cell Reports.

[8]  T. Davis,et al.  Zeatin-induced shoot regeneration from immature chickpea (Cicer arietinum L.) cotyledons , 2004, Plant Cell, Tissue and Organ Culture.

[9]  T. Thorpe,et al.  The role of cotyledonary tissue in the differentiation of shoots and roots from cotyledon explants of Brassica juncea (L.) Czern. , 2004, Plant Cell, Tissue and Organ Culture.

[10]  P. K. Jaiwal,et al.  In Vitro Regeneration and Genetic Transformation of Chickpea , 2003 .

[11]  R. Ortiz,et al.  Program for the application of genetic transformation for crop improvement in the semi-arid tropics , 2000, In Vitro Cellular & Developmental Biology - Plant.

[12]  A. Sagare,et al.  Agrobacterium mediated transformation of chickpea (Cicer arietinum L.) embryo axes , 2000, Plant Cell Reports.

[13]  D. Amla,et al.  DIRECT SHOOT REGENERATION IN CHICKPEA (CICER ARIETINUM L.) , 1998 .

[14]  R. Vashishat,et al.  MULTIPLE SHOOTS FROM COTYLEDONARY NODE EXPLANTS OF NON-NODULATING GENOTYPE(ICC435M) OF CHICKPEA, CICER ARIETINUM L. , 1998 .

[15]  Sanchayita Kar,et al.  Efficient transgenic plant regeneration through Agrobacterium-mediated transformation of Chickpea (Cicer arietinum L.) , 1996 .

[16]  R. Chandra,et al.  Rooting and establishment of in vitro grown shoot tip explants of chickpea (Cicer arietinum L.). , 1996, Indian journal of experimental biology.

[17]  A. Sagare,et al.  Direct somatic embryogenesis from mature embryo axes in chickpea (Cicer arietinum L.) , 1994 .

[18]  V. Chowdhury,et al.  FACTORS AFFECTING PLANT REGENERATION IN CHICKPEA, CICER ARIETINUM L. , 1992 .

[19]  P. Saxena,et al.  Thidiazuron induces high-frequency shoot regeneration in intact seedlings of pea (Pisum sativum), chickpea (Cicer arietinum) and lentil (Lens culinaris) , 1992 .

[20]  J. Preece,et al.  Plant regeneration from leaf explants of Rhododendron ‘P.J.M. Hybrids’ , 1991 .

[21]  W. C. Fooshee,et al.  Thidiazuron Stimulates Basal Bud and Shoot Formation in Alocasia × Chantrieri André , 1990 .

[22]  C. Andre.  Thidiazuron Stimulates Basal Bud and Shoot Formation in Alocasia , 1990 .

[23]  B. G. Rao.  Regeneration from induced embryoids of gram (Cicer arietinum L.). , 1990 .

[24]  V. Chopra,et al.  Regeneration in Chickpea (Cicer arietinum L.) through Somatic Embryogenesis , 1989 .

[25]  B. Briggs,et al.  Micropropagation of azaleas using thidiazuron , 1988 .

[26]  S. Riazuddin,et al.  Establishment of callus-tissue culture and the induction of organogenesis in chickpea. , 1988 .

[27]  K. Singh,et al.  Chickpea: world importance and distribution. , 1987 .

[28]  J. Raven.  Biochemical disposal of excess H+ in growing plants? , 1986 .

[29]  A. Chèvre,et al.  In vitro vegetative multiplication of chestnut , 1983 .

[30]  R. Mateles,et al.  Nitrogen metabolism in plant cell suspension cultures: I. Effect of amino acids on growth. , 1975, Plant physiology.

[31]  F. Skoog,et al.  A revised medium for rapid growth and bio assays with tobacco tissue cultures , 1962 .