Sridevy Sriskandarajah

Regenerative Capacity of Cacti Schlumbergera and Rhipsalidopsis in Relation to Endogenous Phytohormones, Cytokinin Oxidase/Dehydrogenase, and Peroxidase Activities

The recalcitrant nature and increased regenerative capacity in relation to in vitro subcultures in two cactus genera Rhipsalidopsis (Easter cactus) and Schlumbergera (Christmas cactus) were studied by examining the endogenous concentrations of several endogenous phytohormones and enzyme activities. Leaf tissue from greenhouse-grown mother plants, in vitro subcultures 1 and 3, and callus tissues were analyzed and correlated with regenerative ability. The cytokinins present in the two cacti genera were mainly isopentenyl-type derivatives. The total content of isopentenyl-type cytokinins in greenhouse-grown leaves of Rhipsalidopsis was more than twice the amount found in greenhouse-grown leaves of Schlumbergera. The total cytokinin content decreased during subculturing. Cytokinin oxidase/dehydrogenase (CKX, EC 1.4.3.18/1.5.99.12) activity increased during subculturing. In Schlumbergera there is no effect of subculturing on CKX and related cytokinin homeostasis. The total peroxidase (POX, EC 1.11.1.7) activity in greenhouse-grown leaves of both genera was low, and the activity increased significantly during subculturing, more specifically in the tissue of Rhipsalidopsis. The results clearly indicated that an enhanced auxin metabolism (biosynthesis, conjugation/deconjugation, and POX activity), in combination with an enhanced CKX activity, shifts the auxin and cytokinin pool, favoring adventitious shoot formation in Rhipsalidopsis, whereas the low level of POX activity, together with auxin autotrophy/conjugation, makes Schlumbergera more recalcitrant.

Agrobacterium tumefaciens-mediated transformation of Campanula carpatica: factors affecting transformation and regeneration of transgenic shoots

An efficient transformation system for Campanula carpatica was developed using Agrobacterium tumefaciens strains LBA4404 (harbouring the plasmid pBI121), and AGL0 (harbouring the plasmid pBEO210). This is the first report on the transformation of C. carpatica. Various factors affecting the transformation efficiency and subsequent regeneration were identified. The age of seedlings from which the explants for transformation studies were taken, and the growth conditions under which the seedlings were grown had a significant influence on the production of transformed shoots. Hypocotyls taken from 12-day-old seedlings grown in the dark were the most productive, with up to 25% of hypocotyls producing transformed shoots. Explants taken from 5-week-old seedlings produced only transformed callus. The medium used for co-cultivation and incubation also had a significant influence on transformation frequency and shoot regeneration. The cultivar “Blue Uniform” was more responsive than “White Uniform”. Both bacterial strains and plasmids were equally effective in producing transformed tissue. Transformed shoots were selected on kanamycin medium, and the presence of the uidA and nptII genes in those selected shoots was confirmed by β-glucuronidase and ELISA analyses, respectively.

Induction of adventitious shoots in vitro in Campanula carpatica

Efficient protocols have been developed to induce adventitious shoots in different types of explants of Campanula carpatica Jacq. More than five shoots per explant developed on hypocotyls of 5-week-old seedlings after 2 weeks of culture. Hypocotyls produced twice as many shoots as the cotyledons. TDZ proved to be about 6 times more efficient than BA. NAA had to be added to the regeneration medium to obtain the optimal balance of auxin and cytokinin to induce shoot regeneration. Significant differences were noted between different growth regulator concentrations in their effects on shoot organogenesis. BA induced double the number of callus clumps as TDZ. Incubation of explants in the dark produced about 6 shoots per explant while those in the light produced about 2 shoots per explant. Explants derived from 5-week-old seedlings were five times more regenerative compared to those derived from 15-week-old seedlings. Explants from cv. White Uniform were more organogenic than those from cv. Blue Clip. Root segments were also found to form shoots when treated with CPPU.

Regeneration from Phylloclade Explants and Callus Cultures of Schlumbergera and Rhipsalidopsis

Phylloclade explants of Schlumbergera and Rhipsalidopsis were cultured in vitro to produce axillary and adventitious shoots. The explants of both species, taken from greenhouse-grown plants, produced only axillary shoots. There was a pronounced improvement in adventitious shoot formation in phylloclade explants of cultivar CB4 of Rhipsalidopsis by increasing numbers of subcultures of axillary shoots used as donor plants. The axillary shoots generated from the explants were either subcultured to produce successive generations of axillary shoot cultures or made into phylloclade explants and tested for adventitious shoot formation at each subculture. The duration of each subculture varied from 6 to 12 weeks. After the first subculture, sporadic adventitious shoot formation began, and after the third subculture 87% explants of cultivar CB4 produced adventitious shoots at a frequency of about 12 shoots per explant. In contrast, there was no improvement in regenerative ability in explants of cultivar Thor-Olga of Schlumbergera up to third subculture. Adventitious shoots could be produced by callus culture too. Cultivar CB4 was highly regenerative, producing as many as 10 adventitious shoots per square cm of callus. In vitro grown plantlets, when transferred to pots continued to show prolific growth.

Transformation of Hibiscus rosa-sinensis L. by Agrobacterium rhizogenes

Summary Transgenic hairy roots were induced from leaf, petiole, and shoot explants of in vitro-raised plantlets of potted Hibiscus rosa-sinensis L. ‘Cassiopeia Wind Yellow’ plants after co-cultivation with three different wild-type strains of Agrobacterium rhizogenes. The highest infection rate (56.4%) was obtained in explants inoculated with A. rhizogenes strain ATCC43057, followed by ATCC15834 (44.0%), and NCPPB8196 (34.4%). The highest infection rate (60.5%) was obtained using leaf basal segments, followed by petioles (44.0%), leaf tips (40.0%), and shoots (20%). A total of 102 putative transgenic root lines were produced. The hairy root lines did not grow without applying 0.2 mg l−1 indole-3-butryric acid (IBA) and 0.2 mg l−1 α-naphthaleneacetic acid (NAA). The presence of T-DNA was confirmed in six hairy root lines, and no A. rhizogenes contamination was detected by polymerase chain reaction.