MSc

Black pepper, known as ‘King of Spices’ and ‘black gold’ is the most important and most widely used spice in the world. India ranks first in area and production. As far as the productivity is concerned, India stands the last position. During the last decades, the productivity of black pepper in India is under drastic reduction. One of the major problems for this is the dreaded Phytophthora foot rot disease.
Genetic transformation is one of the biotechnological tools for crop improvement and in planta transformation is a transformation method which effectively and directly targets germ-line tissues. The present study, ‘In planta transformation via pollen tube pathway in Piper nigrum” was undertaken with the objective to improve the tolerance of Piper nigrum (var. P2) to Phytophthora capsicii and the development of detection system for intragenic plants. The pollen tube pathway transformation technique is a new plant transformation method in which the normal distant hybridization existed by the hybridization of DNA segment (Zhou et al., 1983). Pollen grain as a transfer vector provides a new and useful method for the transfer of foreign genes among flowering plants for the inter-species transfer of genes between P. colubrinum and P. nigrum.

P. colubrinum is used as the DNA donor plant. DNA was isolated from its tender leaves using CTAB protocol of Rogers and Bendich (1994). The quantity of DNA ranges from 428 ngµl-1 to 2097 ngµl-1. The OD260/OD280 ranged between 1.80 and 2.07 indicating the good quality of DNA, without any contamination. The pollination was done after incubating the pollen grains of P. nigrum var Panniyur-2 with the DNA of P. colubrinum. The spikelet observed from the parent plant ranged from 40 to 80 per cent and 69.33 per cent of spikes set to maturity. The average number of seeds per spike was 25.
Embryo Rescue from the treated spikes was done at three-fourth maturity of the spike. Embryo along with endosperm was inoculated to the ½ MS + 1 mgl-1 BA + mgl-1 IAA + 30g sucrose + 0.1g inositol + 7.5g agar media and the maximum germination observed was 71.57 per cent. The germinated seedlings were allowed to form multiple shoots and then inoculated to elongation media. The elongated shoots were rooted in vitro. Phytophthora capsici were inoculated to liquid Ribiero medium and toxins were produced. The concentrated toxin was added to the medium at the rate of 7.5 per cent (v/v). The elongated shoots were inoculated to the media for root development. The in vitro screening of cultures with the production of roots in the putative transformants was recorded 69.33 percent survival and sixty five per cent of the shoots of in vitro regenerated open pollinated seedlings survived. During in vitro screening the leaf necrosis, death of cultures and reduction in thickness and number of roots were observed (Jin-peng and Mu, 2004; Shylaja, 1996).

The in vitro rooted plantlets were hardened and the percentage of survival during hardening was 80.76 percent for putative transformants and 82.85 per cent for open pollinated seedlings. The hardened plantlets were transferred to potting media and artificial screening was done with culture disc of Phytophthora capsici. Among the putative transformant seedlings, the highest percentage of seedlings (42.15) was in class 2, followed by 29.41 per cent in class1 and 20.58 per cent in class 3.Among the open pollinated seedlings also, the highest percentage of plants in class 2 (40.00) followed by 32 per cent of plants in class1 and 22 per cent in class 5. But the final survival of plants was 39.21 per cent in putative transformants and 35.29 per cent in open pollinated progenies. The putative transformants showed increased resistance to P. capsici compared to control or open pollinated plants. The putative transformant progenies (40) were planted out after in vitro screening and subsequent pathological scoring against Phytophthora foot rot. The F1 putative transformants have shown variation in the level of resistance.
The genomic DNA was isolated from the seedlings of black pepper, Piper nigrum var Panniyur-2, and Piper colubrinum using the mini-prep cTAB protocol (Khan et al., 2004) and RAPD analysis was done. A DNA yield of 120.24 to 771.18 ngµl-1 (12 to 77µg/300 mg leaf tissues) was obtained from the pepper leaf samples by this mini-prep method. The primers OPG 08 and OPA 08 were used for screening of the putative transformants, open pollinated seedlings with respect to P. nigrum and P. colubrinum. The 33.89 per cent of the putative transformants showed variation from the P2 and among the open pollinated progenies, 26.66 percent seedlings showed variation, for the primer OPG 08. Among the putative transformants, the extent of variation in banding pattern observed for the primer OPA 08 was 9 percent and 3 per cent among the open pollinated progenies. But, the polymorphism observed did not strictly relate to the disease resistance in the seedlings.

The putative transformants showed variation in the level of resistance to P. capsici. Whether these plants can show the same resistance in the field needs to be further investigated. More number of primers can be used for screening the putative transformants for the confirmation of transformation and disease resistance. The resistance can be further improved by repetitive pollen tube pathway or in planta transformation as back cross method in traditional breeding. Detection and confirmation of the transgene can be made more reliable by using Southern hybridization or other molecular techniques by using a gene or a construct with known sequence information.