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Enzymatic Isolation of the Components of Female Germ units of Hevea brasiliensis towards the Development of Haploids

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Author Affiliations

  • 1Advance centre for Molecular biology and Biotechnology, Rubber Research Institute of India-686 009, Kerala, INDIA

Int. Res. J. Biological Sci., Volume 4, Issue (3), Pages 44-48, March,10 (2015)

Abstract

Hevea brasiliensis, the sole commercial source of natural rubber, is a perennial tree crop with a long breeding cycle which makes genetic improvement through conventional breeding much time consuming and laborious. Female gametophytic studies leading to gynogenic haploids can be used as an alternative in vitro approach towards crop improvement of Hevea. Female Germ Units (FGU) can be isolated and the cells can be cultured in vitro for the development of haploids as well as homozygous diploids which may be used in further breeding programmes. Also the components of FGU, which are all haploid in nature except the central cell, can be utilized for breeding through in vitro fertilization and gameto-somatic hybridisation. The present study was undertaken to standardise an efficient method for the isolation of FGU from female flowers of Hevea brasiliensis. Ovules isolated from mature female flowers, one day before anthesis, were exposed to different combinations and concentrations of the enzymes cellulase, pectolyase, macerozyme and rhozyme for different time intervals (2-20h). An enzyme solution containing 0.6% cellulase (Onozuka R-10), 0.4% macerozyme (R-10), 0.6M mannitol, 5mM MES, 1g l-1 potassium sulphate and 1g l-l dextrose was found to be the most effective one in the release of FGUs. Egg cell release initiated from the micropylar end of the ovule within 6 h of enzymatic digestion, followed by the release of antipodals from the chalazal end. Maximum release of FGU components was observed by about 16 h of digestion. All the released cells were collected together by centrifuging at 100 rpm for 2 min and cultured in different media for further development.

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