IN VITRO CALLUS PROLIFERATION FROM LEAF EXPLANTS OF GREEN GRAM AFTER IN SITU UV-B EXPOSURE

Callus induction was tried with leaf explants (third leaf from top of canopy) harvested from in situ control and supplementary UV-B irradiated (UV-B = 2 hours daily @ 12.2 kJ m d; ambient = 10 kJ m d) three varieties of green gram viz. CO-8, NVL-585 and VAMBAN-2 to study their viability for germplam conservation. Callus induction occurred both in control and UV-B stressed NVL-585 leaf explants. VAMBAN-2 both in unstressed and UV-B stressed conditions did not initiate callus. Only control leaf explants from CO-8 proliferated callus. Callus of UV-B irradiated NVL-585 weighed less (51.28 %) than control. Parenchyma cells were smaller in callus inducted from in situ UV-B exposed NVL-585 leaf explants. The leaf explants from UV-B stressed NVL-585 varieties of green gram responded to in vitro callus proliferation making them fit for germplasm conservation for cultivating in UV-B elevated environment.


In situ UV-B radiation
Green gram (Vigna radiata (L.) Wilczek.), the nitrogen fixing grain legume was chosen for the study.Viable seeds of the three varieties of green gram viz.CO-8, NVL-585 and VAMBAN-2 were procured from Saravana Farms, Villupuram, Tamil Nadu and from local farmers in Pondicherry.The seeds were selected for uniform colour, size and weight and used in the experiments.The crops were grown in pot culture in the naturally lit greenhouse (day temperature maximum 38 ± 2 ºC, night temperature minimum 18 ± 2 ºC, relative humidity 60 ± 5 %, maximum irradiance (PAR) 1400 μmol m -2 s -1 , photoperiod 12 to 14 h).Supplementary UV-B radiation was provided in UV garden by three UV-B lamps (Philips TL20W/12 Sunlamps, The Netherlands), which were suspended horizontally and wrapped with cellulose diacetate filters (0.076 mm) to filter UV-C radiation (< 280 nm).UV-B exposure was given for 2 h daily from 10:00 to 11:00 and 15:00 to 16:00 starting from the 5 DAS (days after seed germination).Plants received a biologically effective UV-B dose (UV-BBE) of 12.2 kJ m -2 d -1 equivalent to a simulated 20 % ozone depletion at Pondicherry (12º2'N, India).The control plants, grown under natural solar radiation, received UV-BBE 10 kJ m -2 d -1 .Leaf explants (third leaf from top of canopy) were harvested from 30 DAS crops that received supplementary UV-B irradiation and sunlight in the in situ condition.

In vitro culture with leaf explants
Leaf explants after appropriate aseptic treatment were used for in vitro culture.Leaf discs were thoroughly washed with water containing 0.1% Bavistin (a systemic fungicide BASF, India Ltd., Bombay) for 4-5 minutes.They were surface sterilized with 0.1% HgCl2 for 4-5 minutes and washed 6 to 8 times with autoclaved water under Laminar Air Flow Cabinet (Technico Systems, Chennai) and inoculated aseptically onto culture medium.The final wash was given with aqueous sterilized solution of (0.1%) ascorbic acid.The surface sterilized explants were dipped in 90% ethanol for a short period (40 seconds).
The leaf discs were inoculated horizontally on MS medium for culture initiation.Different concentration and combination of cytokinins (6-benzyl amino purine -BAP and Kinetin ranging from 0.1 to 5.0 mg L -1 ) and auxins (IAA -Indole acetic acid ranging from 0.1 to 1.0 mg L -1 ) were incorporated in the medium for inducing callus.These cultures were incubated at 28±2˚C in the dark for 2-3 days.Subsequently these were kept under diffused light (22 µ mol m -2 s -1 SFP-spectral flux photon) for 8 to 10 days.The light was provided by fluorescent tubes and incandescent bulbs.Temperature was maintained by window air conditioners.Positive air pressure was maintained in the culture rooms, in order to regulate temperature and to maintain aseptic conditions.The cultures were regularly monitored and the callus proliferation were recorded after 30 DAI (days after inoculation).The experiments were carried out with three replicates per treatment.
The plant tissue culture media generally comprise of inorganic salts, organic compounds, vitamins, gelling agents like agar-agar.All the components were dissolved in distilled water except growth regulators.Auxins were dissolved in 0.5N NaOH or ethanol and cytokinins were dissolved in dilute 0.1N HCl or NaOH.For the present study MS basal medium (Murashige and Skoog 1962) was used as nutrient medium.
MS basal medium was used either as such or with certain modification in their composition.Sucrose and sugar cubes were added as a source of carbohydrate.The pH of the media was adjusted to 5.8 ± 2 with 0.5N NaOH or 0.1N HCl before autoclaving.The medium was poured in the culture vessels.Finally the medium was steam sterilized by autoclaving at 15 psi (pounds per square inch) pressure at 121˚C for 15 minutes.

Chemical composition of MS medium (Murashige and Skoog 1962)
Constituents Quantity (mgL

Preparation of MS medium
Approximately 90 % of the required volume of the deionized-distilled water was measured in a container of double the size of the required volume.Dehydrated medium was added into the water and stirred to dissolve the medium completely.The solution was gently heated to bring the powder into solution.Desired heat stable supplements were added to the medium solution.Deionized-distilled water was added to the medium solution to obtain the final required volume.The pH was adjusted to required level with NaOH or HCl.The medium was finally dispensed into culture vessels.The medium was sterilized by autoclaving at 15 psi at 121˚C for appropriate period of time.

Photography
The anatomical features were viewed through Nikon Labomed microscope under incident and translucent light and photographed using Sony digital camera fitted with Olympus adaptor.The culture tubes with leaf explants and callus were photographed in daylight using a Sony digital camera fitted with appropriate close-up accessories.

Dendrogram
At least three replicates were maintained for all treatments and control.The experiments were repeated to confirm the trends.The result of single linkage clustering (Maskay 1998) was displayed graphically in the form of a diagram called dendrogram (Everstt 1985).The similarity indices between the three varieties of green gram under study were calculated using the formula given by Bhat and Kudesia (2011).
Similarity Index = Total number of similar characters Total number of characters studied × 100 Based on the similarity indices between the three varieties of green gram, dendrogram was drawn to derive the interrelationship between them and presented in Table 2 and Plate 5.

1
) and auxins (IAA -Indole acetic acid = 1.0 mgL -1 ) was found to be best suited for initiating callus in leaf explants (Plate 1) and used for leaf explants harvested from all varieties of green gram (Plate 2).

In vitro callus induction
In leaf explants proliferation of callus occurred only in two green gram varieties out of the three varieties taken for study (Table 1; Plate 2, 3).Callus induction was observed in control leaf explants of CO-8 and both in control leaf explants as well as in leaf explants harvested from in situ supplementary UV-B irradiated NVL-585 crops.However, all leaf explants from VAMBAN-2 failed to induct callus.The induction of callus was delayed by one day in explants harvested from in situ UV-B irradiated NVL-585 compared with the control (Table 1).However, in CO-8 callus was inducted one day earlier to NVL-585 control.
The callus of in situ UV-B stressed NVL-585 variety of green gram weighed less by 51.28 % compared to control.The control CO-8 had better callus as evident by an accumulation in the fresh biomass by 10 % over the control callus of NVL-585.The trend observed in fresh weight continued in dry weight of callus also.The callus of in situ UV-B irradiated NVL-585 variety weighed less by 78.42 % below control on 30 DAI, while CO-8 control recorded the maximum (Table 1).The parenchyma cells of calluses proliferated from leaf explants were isodiametric with thin cell walls and were distributed evenly all through the callus in control samples (Plate 4, Fig. 1, 2).The parenchyma cells that have proliferated from the in situ UV-B irradiated callus were smaller by 29.67 % and more in number by 17 % in NVL-585 than their control (Plate 4, Fig. 2, 3).This is in accordance with the findings of Rajendiran et al. (2014a) who have reported the failure of leaf explants of some of the varieties of cowpea to proliferate callus after ultraviolet-B irradiation under in vitro culture.Rajendiran et al. (2014b) and Rajendiran et al. (2014c) opine that the response shown by stem explants and seeds to in vitro culture differ based on the sensitivity of the crops to in vitro culture conditions.Rajendiran et al. (2015a) in Amaranthus dubius Mart.Ex.Thell., Rajendiran et al. (2015e) in Macrotyloma uniflorum (Lam.)Verdc., Rajendiran et al. (2015f) in Momordica charantia L., Rajendiran et al. (2015g) in Spinacia oleracea L., Rajendiran et al. (2015h) in Trigonella foenum-graecum (L.) Ser., Rajendiran et al. (2015i)

Conclusion
From the present experiment it is evident that the leaves of NVL-585 variety of green gram are the appropriate explants for germplasm conservation for surviving in UV-B elevated environment, as they are the only plant material to respond to in vitro culture after UV-B irradiation.

Plate 1 :
Standardisation of Kinetin (K) concentration in culture media for in vitro regeneration from leaf explants using Vigna radiata (L.) Wilczek var.NVL-585 control samples.(7 DAI -Days after inoculation)

Plate 3 :
A closer view of calluses formed in two out of three varieties of Vigna radiata (L.) Wilczek from leaf explants of control and UV-B irradiated plants.Plate 4: Cross section of calluses formed in two out of three varieties of Vigna radiata (L.) Wilczek from leaf explants of control and UV-B irradiated plants.(All figs.400x) Dendrogram The variation shown by leaf explants of three varieties of green gram after in situ supplementary UV-B radiation in the parameters viz., time taken for callus initiation, fresh and dry weight of callus, frequency and size of parenchyma cells in callus was reflected in the dendrogram.The two varieties viz., CO-8 and NVL-585 had 42.86 % similarity and they formed one group as their leaf explants harvested from either control or UV-B stressed crops or both proliferated callus.VAMBAN-2 remained alone in the cluster showing very little similarity (14.29 %) with other two varieties, as both control and UV-B stressed explants failed to induct callus (Table 2; Plate 5).

Plate 5 :
Dendrogram showing the interrelationship between the three varieties of Vigna radiata (L.) Wilczek in callus proliferation from leaf discs of control and supplementary UV-B irradiated plants -In vitro.

Table 1 :
Characteristics of callus proliferation in leaf explants of three varieties of 30 DAI Vigna radiata (L.) Wilczek from control and supplementary UV-B exposed conditions -In vitro.

Table 2 :
The similarity indices in callus proliferation from leaf explants of three varieties of Vigna radiata (L.) Wilczek after supplementary UV-B exposure -In vitro.
Plate 2: Comparison of in vitro callus proliferation from leaf explants of three varieties of Vigna radiata (L.) Wilczek on 30 DAI (Days after inoculation).