Indian Journal of Biotechnology Vol. 3, January 2004, pp. 124-127 Identification of in vitro responsive immature embryo size for plant regeneration in Sudan grass (Sorghum sudanenses Piper) Sanjay Gupta 1 *, V K Khanna 2 , Rameshwar Singh 2 and G K Garg 3 1 Division of Plant Breeding, ICAR Research Complex for NEH Region, Barapani (Umiam) Meghalaya 793 103, India 2 Department of Genetics & Plant Breeding, 3 Department of Molecular Biology & Genetic Engineering, G B Pant University of Agriculture & Technology, Pantnagar 263 145, India Received 12 February 2002; accepted 28 March 2003 Immature embryos of various sizes from six genotypes of sudan grass (Sorghum sudanenses Piper) were cultured to identify the best in vitro responsive embryo size. The immature embryos size influenced callus formation (days to callus initiation, callus induction frequency, callus growth) and plant regeneration (shoot induction frequency, shoots per callus) parameters. Immature embryos (0.7-1.5 mm) were quicker to initiate callus, induced fastly growing callus in higher frequency and regenerated shoots more frequently and intensely than immature embryos (1.6-2.5 mm) because of the initiation of callus from rapidly dividing scuttelar cells. Frequent germination and initiation of non-embryogenic callus from plumule or radicle were the main reasons for poor callus induction and plant regeneration responses from 1.6-2.5 mm size immature embryos. Thus immature embryos of 0.7-1.5 mm size may be used for embryogenic culture initiation and plant regeneration. Keywords: sorghum, Sorghum sudanenses, immature embryos, immature embryo size, callus induction, plant regeneration Introduction The gems Sorghum includes many species which are source of grain, fibre, fuel and secondary products 1 . S sudanenses, commonly known as Sudan grass, is one of the important species of sorghum, which is primarily used as feed and fodder for animals. It crosses freely with S. bicolor and represents as an important source of germplasm for introgression of useful genes into S. bicolor. Immature embryo explant, used in maize 2 , has become the most widely used explant in cereal tissue culture. In S. bicolor, there are a number of reports describing plant regeneration from immature embryo explant, the size of which influences plant regeneration 3-8 . Plant regeneration from the tissue cultures of S. sudanenses has been reported 7, 9 , but the influence of the size of immature embryos on in vitro plant regeneration in this species has not been assessed so far. Culturing the immature embryos after certain specific days after pollination 6 , measuring the length of immature embryos 7 and judging the endosperm consistency by squeezing the grain 8 are the three ways which have been used by various workers to standardize the appropriate in vitro responsive stage of immature embryos of S. bicolor. In the present investigation, the immature embryos were aseptically measured prior to their inoculation on to the culture media to determine their appropriate size. Materials and Methods Plant Material and Culture Conditions Immature caryopses of six genotypes (SDSL92101, SDSL92102, SDSL92111, SDSL92112, SDSL92115, SDSL92140) from field grown plants of sudan grass were cleansed with 5% Teepol (v/v) detergent solution and surface sterilized in 0.1% solution of mercuric chloride for 2 min followed by 3-4 rinses in sterile distilled water. Immature embryos were aseptically measured, classified into 4 embryo size classes (0.7-1.0, 1.1-1.5, 1.6-2.0 and 2.1-2.5 mm) and cultured onto MS medium supplemented with 2.0 mg/l 2,4-D and 0.5 mg/l kinetin for callus induction. The cultures were incubated in dark at 25 ± 2 °C. The first sub culturing was made after 4 weeks to the same medium on which callus was induced. The number of days from the date of inoculation of explant on the callusing medium to the date of callus visibility was recorded. The callus induction frequency was recorded after 21 days of inoculation as percentage of total number of inoculated explants producing callus. The callus growth was recorded on a visual scale from 1 (very poor callus growth) to 4 (profuse callus __________ *Author for correspondence: E-mail: sanju_in@rediffmail.com