Ex Situ Conservation of an Endangered Fern Elaphoglossum Nilgiricum (Krajina Ex Sledge and Bonner) T. Moore Ex Alston & Bonner using In Vitro Spore Culture

    

    

    Figure 1: In vitro propagation of Elaphoglossum nilgiricum.
A - Spore with Sori; B - Young gametophyte of E. nilgiricum; C - Matured gametophyte of E. nilgiricum in KC Basal Solid medium; D - Matured gametophyte of
E. nilgiricum in KC Basal Liquid medium supplmented with sucrose; E - Female Sex organ of E. nilgiricum; F- Male Sex organ of E. nilgiricum; G - Sporophyte
proliferation of E. nilgiricum; H - 25 days old sporophytes of E. nilgiricum; I - Hadened sporophytes of E. nilgiricum.
    
    

Materials and Methods

Matured fronds of Elaphoglossum nilgiricum (Krajina Ex Sledge and Bonner) T. Moore Ex Alston & Bonner were collected from Kodaikanal Botanic Garden, St. Xavier’s College, Eettipallum, Tamil Nadu. The fronds were washed in running tap water for few minutes. The fronds were cut into small pieces and dried over white absorbent paper at room temperature (25°C). After drying the fronds at room temperature for 24 hrs, the liberated spores were passed through 40 μm nylon mesh to remove the sporangial wall materials and clean spores were collected and stored in refrigerator at 5°C.

The spores were surface sterilized with 0.1% HgCl2 solution for 5-15 min and washed with sterile distilled water for 15-30 min. The surface sterilized spores were inoculated in to different media viz. KC, KN, MS and Mitra [27-30] devoid of sugar and plant growth regulators using sterile Pasteur pipettes and incubated at 25°C ± 2°C under 12 h photoperiod (1500 lux). The pH of the media was adjusted to 5.8 before adding agar 0.5 % (w/v) and autoclaved at 121°C for 15 min. Both liquid and agar nutrient media were used for spore germination and sporophyte formation. Gametophytes regenerated from spores were sub-cultured on different basal media (KN, KC, Mi, MS and ½ MS medium) for sporophyte formation. Germination percentage of the spores and their development pattern were examined. Photomicrographs were taken with an Optika microscope. The culture tubes containing spore raised micropropagated plants of Elaphoglossum nilgiricum were kept at room temperature (30-32°C) for a week before transplantations. For acclimatization, the plants with well developed rhizoids (5-8 cms) were removed from culture tubes, washed in running tap water to remove the remnants of agar and each group was planted separately onto 10 cm diameter polycup filled with different potting mixtures river coconut husk; garden soil and farm yard manures (1:1:1). The plants were kept in mist chamber with a relative humidity of 70%. Plants were irrigated at 8 h intervals for 3-4 weeks and establishment rate was recorded. The plantlets established in community pots were transferred to shade net house for 3-4 weeks and then repotted in larger pots (20 cm diameter) with one plant in each pot.

Results and Discussion

The spores were sterilized with 0.1% (w/v) mercuric chloride for 10 min. The sterilized spores were washed with sterile distilled water for 20 min. The optimum concentration of mercuric chloride treatment (0.1%) continued existence of spores and low percentage of microbial contamination. The sterilized spores were inoculated onto various media for germination. After 86 days, the spore germination was noticed in KC Basal solid medium. The highest percentage of spore germination (86 ± 3.65) was observed in KC basal agar medium. The effect of medium on spore germination, gametophyte formation and sporophyte proliferations were illustrated in Table 1. Other tested mediums are failed to show the spore germination. Similar to the present observation Manickam et al. [15] also observed the highest percentage of spore germination in KC basal medium for Cheilanthes viridis. Archana et al. [31] also attempted to germinate the spores of Diplazium esculentum in Knop’s, Knudson and Murashige and Skoog’s medium. They observed the highest percentage germination in the Knop’s medium. Even though the Diplazium esculentum and Elaphoglossum nilgiricum belongs to the same group Aspidales they require unique nutritional condition for their growth and development. Bonomo et al. [32] observed spore germination of Alsophila odonelliana in all three studied nutritive (Dyer, KN and KC) medium. The result suggested that the universal media may not be employed for the spore germination of ferns. The spores require specific nutritional environment for germination and growth and development of ferns. To know the importance of nutritional composition on the growth and development of gametophyte, the spore derived prothalli were sub cultured on to fresh medium viz., KN, KC, Mi, MS and ½ MS basal medium. The spore derived gametophytes bestowed extensive gametophyte multiplication and sex organ development in the studied medium (Table 1). The appearance of female sex organs was observed on 41st day from the germination. The cordate gametophyte showed male sex organs on 67th day of germination. The appearance of sex organs on the gametophytes E. nilgiricum was noticed and results were recorded. The gametophytes bearing with sex organs were sub cultured in to the fresh nutrient basal medium (solid and liquid) for sporophyte proliferation. The basal media were failed to support the proliferation of E. nilgiricum sporophytes. The KC liquid medium supplemented with 1.5% of sucrose illustrated highest percentage (56.66 ± 4.08) of sporophyte proliferation followed by KN (48.66 ± 3.80) combination with 1% of sucrose. Similar to the present observation, Johnson and Manickam [33] also witnessed highest percentage of Cheilanthes viridis sporophyte emergence in KC nutrient medium supplemented with 1% sucrose.

  

    
Medium 
    
% of Spore    Germination 
    
% of Gametophyte    multiplication 
    
% of Sporophyte    formation 
    
% of Sporophyte    established in KBG 
  
  

    
KC basal
    
86 ± 3.65
    
86 ± 2.78
    
-
    
-
  
  

    
KC liquid
    
-
    
91.33 ± 1.82
    
56.66 ± 4.08
    
72 ± 4.47
  
  

    
KN basal
    
-
    
86.66 ± 2.35
    
-
    
-
  
  

    
KN liquid
    
-
    
95.33 ± 2.98
    
48.66 ± 3.80
    
-
  
  

    
Mitra basal
    
-
    
85.33 ± 2.98
    
-
    
-
  
  

    
Mitra liquid
    
-
    
89.33 ± 2.78
    
-
    
-
  
  

    
MS basal
    
-
    
80.66 ± 2.98
    
-
    
-
  
  

    
MS liquid
    
-
    
87.33 ± 2.78
    
-
    
-
  
  

    
½ MS basal
    
-
    
83.33 ± 2.35
    
-
    
-
  

Table 1:  Effect of medium on spore germination of E. nilgiricum.