CYTOMORPHOLOGICAL STUDIES IN INDUCED POLYPLOIDY OF LABLAB PURPUREUS L.VAR. TYPICUS PRAIN.

Meenakshi Dheer (Molecularbiotech333@gmail.com)
Department of Botany, University of Rajasthan, Jaipur
April, 2013
 
PhD Scholar, Department of Botany, University of Rajasthan, Jaipur
 

Abstract
The present investigation entitled “Cytomorphological studies in induced polyploids of Lablab purpureus syn. Dolichos lablab L. var. typicus Prain” was conducted at department of Botany, Rajasthan University, Jaipur during (2006-2009) and (2010-2012). The pure seeds of field bean (Lablab purpureus L. Sweet var. typicus prain) were used in the present investigation. Three methods viz. seed treatment, seedling treatment and both seed and seedling treatment had been implemented for inducing polyploidy in Dolichos lablab L. var. typicus, using different concentration of colchicine.
The findings conclude that seeds treated with colchicine had negative impact on seed germination. Thus seed treatment with colchicine was not successful method for the induction of polyploidy in Dolichos lablab L. var. typicus. The chief cause of failure appeared to be the drastic effect of the drug on roots, which failed to produce lateral roots or to show any appreciable development after treatments. After failure of seed treatment, another method seedling treatment with colchicine using cotton swab was followed for the induction of polyploidy in Dolichos lablab L. var. typicus. Plants were treated with different concentration and duration of colchicine i.e. from 0.2 and 0.3% colchicine for 6 hrs., 9 and 12 hrs duration, and 0.4 and 0.5% colchicine for 6 and 9 hrs duration. In seedling treatment method colchicine was added drop wise on the apical tip of the plant with the help of cotton swab and syringe at cotyledonary stage.
Fifty plants were used for each concentration and duration of treatment. In C1 generation morphologically variable plants among the treated plants were selected and tagged. Different morphological variable data were recorded and analyzed statically. Different type of morphological variation in, plant, stem, leaf, flower, pod and seed were recorded. In C1 generation four different plant types viz. dwarf, giant, climber and bushy were observed. Generally plant height decreased with the increase in concentration of colchicine. However plants were, giant in size when treated with 0.2 and 0.3 % colchicine for 9 and 12 hrs of duration and bushy when treated with 0.2 and 0.3 % colchicine for 6 hrs. Climbers were also observed when treated with 0.2 % colchicine for 6 and 12 hrs of duration.Plant suspected as tetraploid was observed when seedlings were treated with 0.5 % colchicine for 6 and 9 hrs. duration. The height of such plant was less (29.00 ± 0.85) than diploid plant (71.68 ± 0.72). Stem diameter also increased at higher concentration and duration of colchicine treatments. The maximum 12 % and minimum 6 % value of thick stem plant was calculated when seedlings were treated with 0.5% colchicine for 6hrs. duration and 0.2 % colchicine for 9hrs. duration, respectively. In suspected tetraploid plant the stem diameter was more (4.40 ± 0.62) than diploid (3.40 ± 0.16) plant.
Treatment with Different concentration and duration of colchicine altered the normal branching (alternate) to zig- zag and basal branching pattern. Maximum percentage (14% plants) of zig-zag branching was observed only with 0.2 % colchicine treatment for 6 hrs duration, whereas basal branching was observed with all the treatments in range of 10 - 14 %. In suspected tetraploid plant branching pattern was basal. The mean number of nodes per branch was also reduced in most of the treatments. In suspected tetraploid plant, mean number of nodes per branch was 10.50 ± 1.41 as compare to 30.20 ± 0.44 in diploid plant.
Treatment of colchicine affected the leaf shape, size, serration, colour and texture in C1 generation. In treated plants leaves were altered, dark green, leathery, deeply serrated. Highest number of leaf variants (89) were recorded with 0.4 % colchicine treatments (6 hrs. duration), while lowest value of leaf variants was observed with 0.2 % colchicine treatments for 6 hrs duration. In suspected tetraploid plant number of branches/plant, number of leaves/plant, length of petiole (cm), length of odd and even leaflet was 35.00 ± 0.89, 75.00 ± 0.13, 6.20 ± 1.24, and 5.60 ± 0.47 respectively. In contrast to these values were higher in diploid plant and was 47.00 ± 0.95, 141.00 ± 1.18, 11.9 ± 0.84, 3.98 ± 0.28 and 0.40 ± 0.06 respectively. In suspected tetraploid plant leaf was dark green, leathery, deeply serrated, and of altered shaped. Rough, leathery and shiny textured of leaves increased along with the increase in concentration and duration of colchicine treatments.
Colchicine affected the normal morphology of flower. Maximum flower variants (15) were found at 0.2 % colchicine treatment for 12 hrs. duration and minimum (6) floral variants were observed at 0.3 % colchicine treatment for 6 hrs duration. Flower size and leathery texture increases with the increase concentration and duration of colchicine treatment. In suspected tetraploid plant flower was terminal in position, leathery in texture and larger (7.01 ± 0.724), than diploid plant where flower size was (5.90 ± 0.51). In suspected tetraploid plant number of flowers per inflorescence was 4.80 ± 0.24 as compare to 9.20 ± 0.49 in diploid plant.
Pod variants were normally observed with higher dose of colchicine. Maximum number (41) of pod variant was obtained with 0.3 % colchicine (9hrs. duration) followed by 35 variant with 0.5 % (9 hrs duration) and 30 variant each with 0.4 % (6 hrs duration) and 0.5 % (6 hrs duration) of colchicine treatment. While in rest of the treatments, these variants were not observed. Pod size reduced with the increase in concentration of colchicine. In suspected tetraploid plant pod were flat, curved, dark green, and rough. The pod size was 4.10 ± 0.51 in treated plant as compare to 3.90 ± 0.31 in diploid plant.
Seed variants were generated at higher dose of colchicine treatments. Maximum (Seed shape, size, colour) seed variants (12) was observed with 0.4% colchicine treatment for 6 hrs duration followed by 11 seed variants each with 0.5 % for 6 and 9 hrs duration and 9 with 0.4 % colchicine treatment for 9 hrs duration. Number of seeds/pod in suspected tetraploid was 4.10 ± 0.15, whereas in diploid plant it was 6.60 ± 0. 25. In suspected tetraploid plant seeds were round, altered, dark and light brown in colour and larger (0.71 ± 0.72) than diploid (0.53 ±.018).Number of seeds per plant (16.00 ± 0.87) was less in suspected tetraploid than diploid plant (125.0 ± 0.51).Highest guard cell index and highest mean values (19.80), (17.20) and (15.14) for guard cell index, was observed in 0.2 % (6 hrs), 0.2 % (9 & 12 hrs). 0.3 % (6 hrs) colchicine respectively. Lowest mean value (6.80) for the same was depicted by 0.5% colchicine treatment (9 hrs duration).
In suspected tetraploid plant number of epidermal cell (22.90  1.09), epidermal cell length (19.40 ± 0.31µm), stomatal length (6.80 ± 0.37) increased by increasing the concentration and duration of colchicine treatment. The number of stomata in suspected tetraploid reduced to 12.20± 0.37 from 35.90 ± 0.44 in diploids at higher concentration. Stomatal studies showed that increased concentration and duration of colchicine treatment increased wavy behavior of epidermal cell. Maximum mean value for stomata length (6.80 µm) was depicted by treatment 0.5 % colchicine (9 hrs. duration). Paracytic type of stomata was observed in all treatments expect 0.2 % (12hrs) and 0.3 % (6hrs) colchicine treatment. Diacytic type of stomata was found at 0.2 % (9hrs duration) and above concentration of colchicine, while anomosytic type of stomata was observed in 0.2 and 0.5 % (6hrs duration) and 0.4 % (9hrs duration) concentration and duration of colchicine. Stomata of suspected tetraploid were large and diacytic type with altered and wavy epidermal cell wall whereas stomata of diploid were anomocytic, diacytic and paracytic types with straight epidermal cell. Maximum mean value of stomatal index (39.00) was investigated with 0.2 % (6 hrs). Whereas, minimum mean value (13.10) for the same was expressed by 0.5 % colchicine treatment (9 hrs).
In suspected tetraploid plant stomatal index (13.10 ± 0.48), guard cell index (6.80 ± 1.73), pore area index (129.00 ± 1.63) were lesser than diploid plant (42.18 ± 0.09, 21.73 ±1.17, 153.00 ± 1.06), while total pore index (0.26 ± 0.003) in suspected tetraploid plant was higher than diploid plant (0.07 ± 0.48).Highest mean value (131.30) for pore area index was found with colchicine dose 0.5% (9 hrs) followed by 129.10 at 0.5% (6 hrs) and 119.00 at 0.4% (9 hrs). It was minimum (97.60) at 0.2% (6 hrs) of colchicine treatment.Maximum mean value of total pore index (0.30) was shown by treatment 0.2% (12 hrs duration), whiles minimum (0.01) by 0.2% (6 hrs) and 0.3% (6, 9 hrs) of colchicine treatment.
Buds of morphological variable plants were collected between 4.00am- 5.00 am for the cytological studies. After the cytological studies four suspected tetraploid plant was found in 0.5 % colchicine treatment for 6 and 9 hrs. duration. Plants of other concentration and duration showed meiotic abnormality. Multinuclei formation was observed at 0.2 % (9, 12hrs), 0.3% (9, 12 hrs),0.4% (6, 9 hrs) and 0.5% (6, 9 hrs) concentration and duration of colchicine treatment. Suspected tetraploid plants were obtained in 0.5 % (6,9hrs) treatments. Suspected tetraploid plants showed doubling of chromosome from (2x=24) in diploid to (4x=48) in tetraploid at metaphase and diakinesis phase. Bivalents were higher than tetravalents in suspected tetraploid plants. Chiasmata were not apparently seen. Equal separation (24:24) and unequal separation (24:23) with laggards was observed during anaphasic separation.
Pollen viability decreased generally with the increase in concentration of colchicine. Pollen shape and size varies in different concentration and duration of treatment. Viable, uneven and unequal size of pollen was observed at 0.2% colchicine treatment for 6 hrs. duration while altered and viable pollen were observed in 0.2% colchicine ( 9,12 hrs duration) treatment as well as in 0.3% colchicine treatment for 6 hrs., 9 and 12 hrs duration. At 0.4% colchicine treatment for 9, 12 hrs duration, pollen was altered shaped and unequal in size. In treated plant maximum number of pollen fertility was observed in 0.2 % concentration of colchicine at 6 hrs duration of treatment while minimum in 0.5% colchicine at 9 hrs duration of treatment. i.e. 92.18 ± 0.03 and 48.58 ± 0.58 respectively. Size of pollen grain and tetrad was maximum in 0.5 % at 9 hrs duration of treatment i.e. (12.8 ± 0.37 x 13.4 ± 0.24, 12.99 ± 0.23×14.87 ± 0.23) and minimum in diploid plant (9.2 ± 0.37x5.4 ± 0.24, 8.9 ± 0.37×12.1 ± 7.12). At different concentration and duration of treatment tetrads showed slight variation, While in suspected tetraploid pollen grain and tetrads were larger (11.2 ± 0.63×15.2 ± 0.77) than diploid (9.2± 0.83×14.2 ± 0.83). The Pollen fertility was 97.1 ± 12.87 % in diploid as compare to 34.3 ± 13.7 in tetraploids plants. Diad, triad, polyyad type of tetrad were observed in suspected tetraploid plant.
Crossing has been done in between diploids and relatively fertile suspected tetraploids. Eight seeds were obtained from one hundred crosses. Six seeds were deformed and failed to germinate. Out of remaining two seeds, one died after germination and another showed little flowering with few flowers. Cytological studies could not be performed due to lack of sufficient buds.Ten seeds from four suspected tetraploid plant collected in C1 generation was sown for the next C2 generation. Five seeds were shrunken and did not germinate, but other five germinated. Out of five plants one plant was suspected triploid and another plant was suspected monosomic. Rest of plants was diploids in C2 generation.
Quantitative and qualitative traits of suspected triploid, monosomic and morphologically variable plants were studied. Seeds from suspected monosomic and triploid plants were collected, which failed to germinate.Plants of C2 generation were dwarf, light green in colour with shiny leaves and large pods. The height was maximum (54.87 cm.) and minimum (46.01 cm) in 0.2 % colchicine (6 hrs. treatment) and 0.5 % colchicine (9 hrs. treatment) respectively. Stem of plants was weak and light green in colour. Maximum stem diameter 2.94 cm. was measured at 0.5 % concentration of colchicine (6 hrs. treatment) followed by 2.93 and 2.92 cm at treatment of 0.4 % concentration of colchicine for 6 and 9 hrs respectively. Maximum pod length (7.90cm) was measured at 0.2 % colchicine (6hrs treatment) and minimum (5.78cm) at 0.5% colchicine (9hrs treatment). Maximum seed size (0.38cm) was found at 0.2 % colchicine (9hrs treatment), while minimum (0.21cm.) at 0.5% colchicine (5hrs treatment).
Two variable plants were obtained from the seeds of suspected tetraploid. These plants were suspected as triploid and monosomic plant. Morphological characters of suspected triploid plant were similar to triploid plants. Suspected tetraploid plant had reduced flower and fruit setting, and seeds were altered, shrunken, large and light brown in colour and failed to germinate. Flower size was slightly larger than control. Pollen sterility was high in this plant. During cytological observations, in diakinesis, 3x=36 chromosome were observed. Chiasmata ring and rods formation was not clear. Suspected monosomic plant was week, light green in colour; pods and seeds were altered, flat or round. Cytologically (2n-1=23) chromosome were observed at metaphasic plate. Anaphasic separation and diakinesis was not found.