41

The New Plant Type (NPT) with distinct morphological characters, such as thick and sturdy stems, dark green thick erect leaves, low numbers of tillers, large panicles with high number of grains, high harvest index (0.6) and short stature (90-100 cm), has been developed to increase the yield potential of rice by 20-25% (Khush 1995). However, additional breeding work is underway to incorporate genes for disease and insect resistance into these lines. The successful introduction of gus and hph genes into NPT lines through transformation indicated the possibility of introducing agronomically important genes into these lines.

Five selected lines of New Plant Type (IR65598-112-2, IR65600-1-2-3, IR65600-42-5-2, IR66738-118-1-2, and IR66743-41-2-2) were used. Both biolistic (Christou et al. 1991) and protoplast (Datta et al. 1990, 1992) methods were used for transformation, Three kinds of explants eg. immature embryos, embryogenic calli and embryo-genic cell suspensions were used for particle bombardment. Isolated protoplast from 1 -3 month old suspensions were used for PEG mediated transformation. Plasmid, pGHi (carrying gus and hph genes) and pGL2 (carrying only hph gene) were used for both methods of transformation (Fig. 1-3).

The results are presented in Table 1. The molecular analysis by Southern blot showed the stable integration of both gus and hph genes into the genomic DNA of NPT lines (data not shown). The integration and function of transgenes hph and gus were confirmend by HPT (hygromycin phosphotransferase) assay of To and T1 plants (HPT-data not shown). Expected Mendelian 3:1 (16:6) segregation ratio for gus expression was observed in T1 plants (Fig. 4). It is advantageous to use husk for gus staining, as the kernels with positive reaction can be grown to maturity. The seeds from the gus* husks showed positive blue staining (arrow indicates gus* seed in Fig. 4A). The fertility of the primary transformants ranged from 70-80%. No abnormal morphological features were observed excepting reduced height. This initial success is encouraging for developing transgenic NPT plants with agronomically important genes such as bt and chitinase. We acknowledge the financial of Rockefeller Foundation and BMZ of Germany. Plasmid DNAs, pGL2 and pGHi were provided by Drs. J. Paszkowski and G. Neuhaus from ETH-Zurich, Switzerland.

Table 1. Transformation of New Plant Type using biolistic and protoplast stystems

PMS-plants at maturing stage, analysis not yet done. So far 10 plants are fertile and inherited gus and hph genes.


No. of experiments	NPT lines	Methods	Genes used	Hg r selected calli	Number of putative transgenic plants regenerated	Molecular analyses by southern blot/ HPT assay
1-3	IR65600-1-2-3	Biolistic	gus+ hph	15	12	12 plants, gus+.* HPT*+
4-5	IR65600-42-5-2	Biolistic	gus+ hph	6	6	6 plants, gus+, HPT+
6	IR66738-118-1-2	Biolistic	hph	5	10	2 plants HPT+ others PMS
7	IR65600-1-2-3	Protoplast by PEG	hph	110	32	32 HPT+
8	IR65600-42-5-2	Protoplast by PEG	hph	112	35	11 HPT+ other PMS