ABSTRACT:The objective of the present study was to understand the relationship among leaf morphology, stomatal characteristics and water relations in triploids generated through anther culture and their counterpart diploid plant of C. clementina. Triploid plants possessed small and narrow leaves as compared to diploid plant as evident by less leaf length, leaf width and leaf area. By contrast, the leaf index was observed to be more in triploids than haploid ones. Flow cytometric analysis re-confirmed the ploidy levels of heterozygous plant Hd as diploid and the ploidy of Th1, Th2, Th3 and Th4 plants as triploids. A positive relation was found between ploidy level and stomatal guard cell length and width, whereas a negative relation was observed between the stomata density and ploidy level. The stomatal density was reported to be 6.2±0.2 stomata per µm 2 in diploid plant, while stomatal density varied between 3.0 and 3.6 stomata per µm 2 in triploids.Leaf relative water content (RWC) was slightly higher in triploids (90.8 to 93.1%) than diploid (89.5%). The leaf water loss was found to be marginally higher in diploid than in triploid plants. Our results show that increase in ploidy level from diploids to triploids caused an effect on leaf morphology and stomatal characteristics with probable consequences to water relations of leaves. This research will serve as an important basis for future work on complete analysis of both morphological and behavioural traits of the leaf stomata and transpiration rates in relation to diploid versus triploid plants.
In vitro clonal multiplication of Pterocarpus santalinus L. was achieved using mature nodal explants of a 10-year-old elite quality tree. Combinations of serial transfer technique and incorporation of antioxidants (250 mg/l l-ascorbic acid and 50 mg/l citric acid) into the culture medium helped to minimize medium browning and improve explant survival during shoot sprouting. About 70% of explants were sprouted on Murashige and Skoog (MS) liquid medium containing 4.4 mM 6-benzyladenine (BA). The explant harvest period also influenced the bud break and shoot sprouting in nodal explants. The combination of 4.4 mM BA and 2.2 mM thidiazuron (TDZ) was found to be the most suitable growth regulator for obtaining the highest percentage of nodal segment sprouting (74%-75%), the number of secondary shoots per primary shoot (two or three), the shoot length (5-6 cm), the number of new nodal segments generated per active explant (four or five), and the multiplication coefficient (3.5) within 6 weeks. Repeated subculturing of nodal explants obtained from shoot cultures enabled continuous production of healthy axillary shoots. At the end of the sixth passage, about 90% of nodal explants produced five or six healthy green shoots, each being about 6.6 cm long with six or seven nodes. Multiplication coefficient was also increased from the first subculture (5.4) to the sixth subculture (8.3). The best rooting response was achieved on solidified half-strength MS medium supplemented with 4.9 mM indole-3-butyric acid (IBA). About 70% of the micropropagated plantlets were established successfully in 20-cm pots containing a mixture of soil and farmyard manure (4 : 1 ratio) and formed new leaflets.
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