No abstract

Indian citrus ringspot virus (ICRSV) is known to cause serious disease problem in Kinnow (Citrus nobilis Lour · C. deliciosa Tenora) plants. This work reports the elimination of ICRSV by using thermotherapy coupled with shoot tip grafting in vitro. Nodal segments from infected mother plants (indexed by indirect ELISA and RT-PCR) were treated both in water bath and moist hot air at different temperatures viz. 40, 45 and 50°C for 30, 60 and 120 min and cultured on MS medium containing 2-iP (1 mg/l) and malt extract (800 mg/l). Shoot tips were excised from the nodal sprouts and grafted on to rough lemon (C. jambhiri) rootstock under aseptic conditions. Water bath treatment was found to be more effective as compared to moist hot air treatment as maximum number of ICRSV free plants (36.84%) were obtained by grafting the tips (0.7 mm) taken from the nodal segments treated at 50°C in water bath for 2 h. In an alternate treatment regime, 1-year-old infected plants were kept at various temperatures viz.36, 38 and 40°C in a thermotherapy chamber. Maximum of 60% ICRSV free plants were obtained by grafting the tips (0.7 mm) from the plants placed at 40°C followed by the plants placed at 38°C (59.09%) and the least was observed in case of the plants placed at 36°C (40.74%). Only those plants/plantlets were considered virus free, which showed negative reaction both in Indirect ELISA and RT-PCR.

The present study deals with the selection of Phytophthora tolerant lines of Citrus jambhiri and their regeneration. Cotyledon derived calli were cultured on selective MS medium supplemented with 5-100 % of culture filtrate (CF), to estimate the critical concentration of the selective agent. The survived calli under stress were subcultured for mass propagation for 20 days on callus multiplication medium (2,4-D 2 mg/L + BA 0.75 mg/L) without culture filtrate. After multiplication, these calli were further exposed to other cycles of selection, which contained the same and the 3 steps higher concentrations of the selective agent (CF) and this procedure was repeated several times until the selection regime completed. The selected tolerant calli were transferred to regeneration medium (MS medium supplemented with 3 mg/L of BA and same concentration of culture filtrate on which the calli were selected). Regenerated shoots were transferred to rooting medium (½ strength MS medium supplemented with 0.5 mg/L of NAA). Under in vivo conditions about 81 % of the selected regenerates exhibited resistance to Phytophthora parasitica, whereas none of the control plants showed resistance.

Citrus jambhiri Lush. (family Rutaceae), commonly known as 'rough lemon', is one of the favourite rootstocks for lemons, oranges, mandarins, grape fruits and kinnows in Punjab. The present investigation deals with development of an efficient miropropagation protocol for Citrus jambhiri Lush. using cotyledons as explant. Maximum callus induction (91.66 %) was observed on MS medium supplemented with 2,4-D (2 mg/L) in combination with ME (500 mg/L). Green healthy calli were cut into small pieces and cultured on MS medium for regeneration. Maximum shoot regeneration (87.50 %) was observed with BA (3 mg/L). Effect of increasing age of callus was also studied which showed that callus retained regeneration capacity (58.33 %) even after 420 days of culture. Regenerated shoots were separated out and cultured on rooting medium. Maximum rooting response (91.67 %) was observed on half strength MS medium supplemented with NAA (0.5 mg/L). After hardening and acclimatization the plantlet were transferred to the field and showed 67 % survival.

Production of Indian citrus ringspot virus (ICRSV)-free plants from an infected plant of kinnow mandarin (Citrus nobilis Lour 9 C. deliciosa Tenora) is reported. The shoot apices of different sizes (0.2-1.0 mm) excised from the ICRSV-infected plant were micrografted onto decapitated rootstock seedlings of rough lemon (C. jambhiri). Micrograft survival depended on the size of shoot apex and the sucrose concentration of the culture medium. Increase in scion size from 0.2 to 0.7 mm resulted in an increase in micrografting success rate from 30.55 to 51.88%. Further, micrograft survival obtained with 0.2 mm was improved from 30.55 to 38.88% by increasing sucrose concentration in the culture media from 5 to 7.5%. The micrografted plants were tested for ICRSV using ELISA and RT-PCR. All plants raised from 0.2-mm scion were found negative with both ELISA and RT-PCR whereas only 20% of the ELISA negative plants raised from 0.3-mm scion were found negative for ICRSV with RT-PCR. The outcome of this research is the successful establishment, acclimatization and virus testing of micrografted plants.

A rapid and highly-effective method for micropropagation from nodal segment and shoot tip explants was established for Coleus blumei Benth. Nodal segments and shoot tips were inoculated on MS medium containing 0.7 % agar, 3 % commercial sugar, and different combinations of 6-benzyladenine (BA) with indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). Hundred percent shoot induction from both explants was achieved on the medium containing BA (2 mg dm -3 ) and NAA (1 mg dm -3 ). Shoot tips were proved to be the better explant in comparison to nodal segments in having high rate of shoot induction and more number of shoots. The same media conditions were found suitable for shoot multiplication. Multiplied shoots rooted best on MS medium supplemented with IBA (2 mg dm -3 ). Micropropagated plants were successfully established in soil after hardening, with 100 % survival rate.

This paper reports the elimination of Indian citrus ringspot virus (ICRSV) from "kinnow" (Citrus nobilis Lour × Citrus deliciosa Tenora) employing phytotherapy coupled with shoot tip grafting under in vitro conditions. Nodal segments from infected mother plant (indexed by indirect enzyme-linked immunosorbent assay [ELISA] and reverse transcriptase PCR [RT-PCR]) were cultured on Murashige and Skoog medium containing 2iP (1 mg/l or 4.9 μM) and malt extract (800 mg/l) along with different concentrations of aqueous extracts from leaves of Azadirachta indica (Neem), Sorghum vulgare (Jowar), and roots of Boerhaavia diffusa (Punarnava). Shoot tips were excised from the nodal sprouts and grafted on to rough lemon (Citrus jambhiri) under aseptic conditions. Maximum effect (50% virus elimination) was seen for aqueous leaf extracts of A. indica followed by B. diffusa root extract (42.86%) and S. vulgare leaf extract (31.58%). Plants/plantlets were considered virus-free only when showing negative reactions by both indirect ELISA and RT-PCR.

The aim of present investigation was to study the effect of storage conditions on percentage germination of encapsulated and non-encapsulated somatic embryos of Kinnow mandarin (Citrus nobilis Lour · C. deliciosa Tenora). Different batches of encapsulated and non-encapsulated embryos were preserved at room temperature, 4°C, in liquid nitrogen as such and by embedding in liquid paraffin. In the encapsulated somatic embryos stored at room temperature in sealed Petri plates, percentage of germination was 24.99%, but 5.55% in non-encapsulated embryos after 3 days of storage. Encapsulated embryos stored in vials containing liquid paraffin at room temperature were germinated at 18.05% after 60 days of storage, while it was 13.88% in non-encapsulated embryos after 45 days of storage. Encapsulated somatic embryos stored at 4°C in sealed Petri plates remained viable for up to 75 days with 6.94% germination, whereas non-encapsulated embryos remained viable for up to 45 days with 24.99% germination. Encapsulated embryos stored at 4°C in vials filled with paraffin germinated at 11.11% after 120 days of storage, but 5.55% in non-encapsulated embryos after 90 days of storage. Encapsulated and nonencapsulated embryos stored in liquid nitrogen showed 58.33 and 51.38% survival, respectively, after 7 months of storage. The plantlets developed from these embryos were transplanted after acclimatization and are growing normal.