Propagation and breeding of kakrol (MOMORDICA DIOICA ROXB.)

Abstract

Prime objective of this study was to establish an effecient method for mass production of propagules of diploid, triploid and tetraploid kakrol (Momordica dioica Roxb.). The induction of somaclonal variation and reproductive behavior of the kakrol were also studied for undertaking future breeding programme. Kakrol cheifly propagates through tuberous root. However, production of tuberous root is low. In addition to this due to hard seed coat, seed germination in kakrol under normal condition, is very low. In this investigation, mass propagation was successfully done in kakrol through treating vine cuttings with 1.0 rngr1 IBA for 30 min. Enhanced rate of seed germination was acheived by removing the seed coat prior to seed sowing. Results of in vitro studies show that, true to type kakrol could also be raised through culture of different explants. Nodal and shoot tip explants of field grown plant rapidly induced multiple shoot in vitro. Among the different media formulations tested 2.0 mgr1 BA + 0.2 mgr1 NAA in MS (Murashige and Skoog, 1962) salt was the best for the induction of multiple shoot from both nodal and shoot tip explant of tetraploid kakrol. Morphological differentiation from juvenile tissues such as cotyledon, embryo and hypocotyl of germinating seeds of tetraploid kakrol in different culture media formulations was investigated. Morphogenic response of cotyledon markedly varied with the ontogenic stage of the explant as well as with growth regulator composition of the media formulations. Cotyledons collected 18-21 days after pollination induced direct multiple proliferation when cultured on MS medium supplemented with 2.0 mgr1 BA + 0.2 mgr1 NAA + 0.1 mgr1 GA3. Callus development occurred from cotyledon and hypocotyl explants in MS medium containing auxin alone or in combination with cytokinin. Growth, morphological nature and organogenic potentiality of the calli varied with explant type and growth regulator supplements. All BA with NAA combinations accentuated organic potentiality of the primary callus. Organic potentiality of cotyledoner callus was more than hypocotyl derived callus. Optimum shoot regeneration occurred from cotyledoner calli when subcultured in 2.0 mgr1 BA+ 0.2 mgl"1 NAA. Whereas, 3.0 mgl"1 BA+ 0.5 mgr1 NAA was optimum for hypocotylar callus. Callus culture was maintained for a long time through subculturing in MS medium containing BA+ NAA or IAA + KIN combinations. Rooting potentiality of the microcuttings varied with their sources of explants. Among the various media compositions tested MS with 1.0 mgr1 IBA medium was the best for root induction. Somaclonal variation m respect of fruit weight among the somaclones regenerated from induced callus was observed. Some of the somaclones had higher fruit weight than normal. Bisexual flowers could be induced in tetraploid, triploid and_ diploid type of kakrol by treating shoots with AgNO3. Most of the AgNO3 treated vines produced continuous female flowers when sprayed with 100 and 200 ppm AgNO3 at 5 days, 7 days or 10 days interval. AgNO3 at 100 ppm on individual twig at 7 days interval produced highest number of normal female flowers in tetraploid type ofkakrol. The application of AgNO3 to female plants developed bisexual flowers. Stamens in induced bisexual flowers developed from the base of style and anther reached just beneath the stigma. AgNO3 at 300 ppm produced the highest number of bisexual flowers per vine in diploid type of kakrol whereas 400 and 500 ppm AgNO3 produced highest number of bisexual flowers per vine in triploid and tetraploid type of kakrol respectively. Morphological studies of the reproductive structures reveal that bract size and position on the peduncle of the bisexual flower were different from those of normal male and female flowers. Most of the induced bisexual flowers were bigger than those of corresponding normal male and female flowers. Leaf area in male and female tetraploid plants were larger than corresponding diploid and triploid. Number of viable pollen in induced bisexual flower was higher in tetraplotd than other types of kakrol. The highest percentage of pollen grain germination was 85% recorded in tetraploid and diploid male flowers when 20% glucose solutions were used for 45 minutes. The pollens of bisexual was as effective as that of male flowers on the normal female flowers. In diploid, percentage of success in crosses with pollens from diploid bisexual flowers was same as found in control. The pollen of induced bisexual flower was not effective on the same or other induced bisexual flowers. However, induced bisexual flower did not develop fruit under self or sib or even cross pollination with normal pollen. Fluorescent microscopic observations reveal that 76% of developing pollen tubes of cf tetraploid were able to enter the stigma but they failed to penetrate further due to tumor formation. When pollen grains of normal tetraploid flower were used to pollinate different types of pistillate flowers, fruit setting was observed only in o tetra xd' tetra andodip + + xo' tetra crosses. Fluorescence microscopy shows that, numerous pollen tubes in both cases were found to develop through the stigmatic papillae to perform fertilization. Pollen graini collected from male and bisexual flowers of triploid failed to give successful crosses with any types of pistillate flowers. Following pollination both localized and diffused callose production was observed on the stigmatic papillae which eventually prevented pollen germination. On the other hand the stigmatic papillae from unpollinated pistils did not produce any kind of callose. The fruit produced through the crossing of the stigmas of diploid or tetraploid with the pollens of induced bisexual flowers were greater in size than the fruits developed through other crosses. Seeds from induced cf pollen x o dip or tetra + crosses developed only female plants that could be used for large scale seed production.