Morphogenetic effect of Chromium GA3, IAA on in-vitro grown Solanum tuberosum L

Abstract

The current work deals with the strategy to produce chromium tolerance with GA3 and IAA in Solanum tuberosum cv. Desiree. Objective of present work was to see the morphogenetic effect of chromium, GA3 and IAA on the root, stem and leaf of Solanum tuberosum. In order to proceed with objectives, micropropagation technique was used. Various levels of chromium (100, 200 ppm of K2Cr2O7 or CrCl3), GA3 (12, 16 and 20 ppm) and IAA (4 and 16 ppm) in the MS medium were selected to treat the in vitro nodel sections of potato. Chromium and plant growth regulators were applied individually and in combinations and their effects were evaluated on the basis of several external and internal morphological features. Data thus obtained were compared with the treated, non-treated plants and among themselves. Potato is an important crop in our country which can bear stress of heavy metals to some extent. However, excess of heavy metal can not be tolerated by Solanum tuberosum. Cr (III) and Cr (VI), in the form of K2Cr2O7 and CrCl3 were applied in the MS medium at the level of 100 and 200 ppm. At each concentration of Cr (III) and Cr (VI), reduction in the root length, diameter and number, fresh and dry weight of root, shoot length and diameter, leaf number and area was observed. As far as root length is concerned, exogenous chromium decreased the length from 8.18 to 5.60 cm and 6.68 cm with applied 100 ppm, while 1.25 and 1.46 cm with exogenous 200 ppm of K2Cr2O7 and CrCl3 respectively), diameter of root was dercreased from 0.22 mm to 0.13 and 0.17 with application of 100 ppm reduction was more wth addition of 200 ppm K2Cr2O7 and CrCl3 i.e., 0.11 and 0.15 mm. Similarly average number of root and rootlets was reduced from 7.5 to 4.78 and 5.20 in the MS medium containing 100 ppm K2Cr2O7 and CrCl3. When level of K2Cr2O7 and CrCl3 was increased number was 1.24 and 1.5. Like wise number of rootlets was dereased. Fresh and dry weight of root was decreased in 100 and 200 ppm K2Cr2O7 or CrCl3 than control plants. Shoot length was reduced from 9.62 cm to 8.15 and 4.53cm with treatment of 100 and 200 ppm K2Cr2O7, similarly reduction was 8.09 and 5.86 cm with 100 and 200 ppm CrCl3. Shoot diameter was decreased in K2Cr2O7 or CrCl3 than control plants. Number and area of leaf with the stress of 100 and 200 ppm K2Cr2O7 or CrCl3 was reduced significantly. K2Cr2O7 proved more toxic than CrCl3 on external morphological features. Toxic effect of K2Cr2O7 or CrCl3 was reduced with applied IAA and GA3. Number of root and rootlets, fresh and dry weight was increased with the application of IAA at the level of 4 and 16 ppm individually. Exogenous IAA increased the width and decreased the length of root. Increase in length of root was recorded with application of GA3 at low concentration. In this study, with application of GA3 (12, 16 and 20 ppm individually) average diameter of roots was reduced. Increase in length was corresponding to decrease in diameter. There was an increase in fresh and dry weight of roots at low concentration of applied GA3. Combination of GA3 and IAA at various levels showed unexpected results may be due complex interaction and crosstalk of these or other plant hormones, which helped for selection of concentrations of GA3 and IAA for mitigating the effect of chromium stress. Shoot length was increased and diameter of shoot decreased with applied GA3. Exogenous GA3 (12, 16 and 20 ppm) alone was used in the present investigation which enhanced the leaf size of S. tuberosum. K2Cr2O7 or CrCl3 along with GA3 and IAA combination improved the length and diameter of adventitious roots, number of root and rootlets, fresh and dry weight of adventitious roots, length and diameter of shoots, number of leaves. Mixed doses of GA3 and IAA at the level of 12 ppm of GA3 + 4 ppm of IAA, 16 ppm of GA3 + 4 ppm IAA and 20 ppm of GA3 + 16 ppm IAA along with K2Cr2O7 or CrCl3 at the level of 100 ppm and 200 ppm were tested. Root length was increased from 5.60 cm to7.57, 7.35 and 7.30 cm with 100 ppm and from 1.25 cm to 2.60, 2.95 and 2.82 cm with 200 ppm of K2Cr2O7 in MS medium containing GA3 and IAA), increase in diameter from 0.13 mm to 0.68 mm with100 ppm K2Cr2O7 along with 20 ppm of GA3 + 16 ppm IAA, number of root (increased from 4.78 to 63.40, 56.64, 62.60 with 100 ppm K2Cr2O7 and from 1.24 to 2.10, 2.00 and 2.40 with 100 ppm K2Cr2O7 ), rootlets (from 2.40 to 13.00, 16.11, 28.50 with 100 ppm of K2Cr2O7 and when K2Cr2O7 stress raised up to 200 ppm the increase was from 0.50 to 1.53,2.50, 1.20), fresh weight (from 34.40 mg to 390.60, 343.00, 1234.74 mg and from 65.05 mg to 80.50, 84.00, 86.20 mg with addition of 100 and 200 ppm of K2Cr2O7 along with GA3 and IAA) and dry weight (10.77 mg to 27.60, 26.23, 143.20 mg) of root. Indicate Shoot length was increased from 8.15 cm up to 13.29 cm, Shoot diameter increased from 0.48 mm to 0.60 mm with 100 ppm of K2Cr2O7, 20 ppm of GA3 and 16 ppm IAA. Numbers of leaves were also increased from 7.70 to 8.40, 9.00, 9.70 with 100 ppm of K2Cr2O7 and 5.50 to 6.40, 6.60, 6.63 with addition of 200 ppm of K2Cr2O7. Leaf area was also increased from 0.72 to .90 cm and from 30 to 0.41 cm 100 and 200 ppm of K2Cr2O7. Data showed inhibition caused by stress of 100 and 200 ppm of K2Cr2O7 can be reversed with GA3 and IAA. Addition of CrCl3 at the level of 100 ppm and 200 ppm along with above mentioned combinations showed partial reversal. For anatomical studies permanent mount were prepared with rotary microtome. Cell size was measured with ocular micrometer. Effect of chromium and plant hormones on epidermal cells, cortical layers, cortical region, cortical cells, width of metaxylem vessels pith cells were studied in transverse and longitudinal planes. Leaf mesophyll was studies in transverse section. All the data with obtained from treated plants was compared with non-treated plants. Applied K2Cr2O7 at the level of 100 ppm and 200 ppm stress affected of length or width of root cells and reduced the length epidermal cells (from 50.52 µm to 33.32, 32.17 µm,), width of epidermal cells (from 13.11 µm to 12.91, 11.58 µm) length of cortical cell (from 44.16 µm to 40.00, 37.73 µm), length of metaxylem vessels (90.38 µm to 88.68, 74.14 µm) width of metaxylem vessels (14.38 µm to 12.60, 10.92 µm).Stem cells were affected by stress of K2Cr2O7 at the level of 100 ppm and 200 ppm length of cells epidermal cells(from 75.79 µm to 70.40, 67.10), width of epidermal cells (from 17.38 µm to 13.50, 12.30) length of cortical cell (from 97.11 µm to 92.80, 81.00), width of cortical cells (25.44 µm to 20.00), width of protoxylem vessels (13.22 µm to 11.50, 8.80) width of metaxylem vessels (25.55 µm to 24.23, 21.08), width of pith cells (33.00 µm to 29.20, 25.05) of stem.Leaf cell showed reduction with applied stress of 100 ppm and 200 ppm K2Cr2O7 i.e., Width of adaxial epidermal cells (22.50 µm to 19.85, 15.67 µm), width of abaxial epidermal cells (21.14µm to 18.09, 15.22 µm), length of palisade cells (47.95 µm to 40.51, 37.87 µm), Width of palisade cells (12.06 µm to11.42, 11.05 µm) and width of spongy mesophyll (20.34 µm to 18.65, 15.42 µm). When MS medium GA3 and IAA along with K2Cr2O7 or CrCl3 increase in above cell length and width was recorded. Out of three hormonal combination 20 ppm of GA3, 16 ppm of IAA increased more length and width of cells along with 100 ppm and 200 ppm K2Cr2O7 or CrCl3. width of root cells and reduced the length epidermal cells (from 33.32 to 35.83, 32.17 to 34.02 µm,), width of epidermal cells (from 12.91 to 14.41, 11.58 to13.52 µm) length of cortical cell (from 40.00 to 42.81, 37.73 to 40.50 µm), length of metaxylem vessels (from 88.68 to 95.87, 74.14 to 78.91 µm) width of metaxylem vessels (12.60 to 15.11, 10.92 to 12.79 µm). Stem cells were affected by stress of K2Cr2O7 at the level of 100 ppm and 200 ppm length of cells epidermal cells (from 70.40 to 73.15, 67.10 to 71.00 µm), width of epidermal cells (from 13.50 to 16. 70, 12.30 to 15.85 µm) length of cortical cell (from 92.80 to 100.05 , 81.00 to 87.35 µm), width of protoxylem vessels (11.50 to 13.83, 8.80 to 10.87 µm) width of metaxylem vessels (24.23 to 27.66 , 21.08 to 22.85 µm), width of pith cells (29.20 to 32.58, 25.05 to 25.89 µm) of stem. Leaf cell showed reduction with applied stress of 100 ppm and 200 ppm K2Cr2O7 i.e., Width of adaxial epidermal cells (19.85 to 20.94, 15.67 to 18.62 µm), width of abaxial epidermal cells (18.09 to 18.80, 15.22 to 17.28 µm), length of palisade cells (40.51 to 41.83, 37.87 to 39.90 µm), Width of palisade cells (11.42 to 14.12, 11.05 to 13.50 µm) and width of spongy mesophyll (18.65 to 20.12, 15.42 to 18.50 µm). However, application of GA3 and IAA along with 100 and 200 ppm K2Cr2O7 or CrCl3 in combinations increased the length, width of cortical cells, width of metaxylem vessels and length of metaxylem vessel members. Hormonal combination of 20 ppm of GA3 + 16 ppm IAA showed more increase in for increasing the width of cortical cells, cortical region, width of metaxylem vessels, length of metaxylem vessel member and width of pith cells. For callus induction, 2, 4- D was used with MS medium. Callus was granular, friable mostly and cream to brown in color. A general decrease in fresh weight of callus was observed with increase in concentration of chromium (CrCl3 or K2Cr2O7). Chromium (K2Cr2O7 or CrCl3) at the level of 100, 200 and 400 ppm proved toxic. Total soluble protein contents and activity of peroxidase (Antioxidant enzyme) of various tissues under stress of K2Cr2O7 and CrCl3 was evaluated to understand the possible role in heavy metal tolerance. In chromium containing mdium a decrease in contents of soluble proteins of the Solanum tuberosum was observed, an increase was registered with applied GA3 and IAA in combinations. Peroxidase activity in stress environment with 100 ppm and 200 ppm of K2Cr2O7 was increased. Hormones were added in the medium along with chromium which increased peroxidase activity in comparison to K2Cr2O7 and CrCl3. However, peroxidase activity decreased with K2Cr2O7 or CrCl3 along with GA3, IAA. In this work, it was found that application of K2Cr2O7 or CrCl3 at 100 and 200 ppm affected seriously to all parameters studied. This reduction was more with applied 200 ppm of K2Cr2O7 or CrCl3 than 100 ppm of K2Cr2O7 or CrCl3. Combination of GA3 and IAA at the level of 20 + 16 ppm along with K2Cr2O7 or CrCl3 at the level of 100 ppm or 200 ppm proved better for reversal than the other combination of GA3 and IAA (12 + 4 ppm, 16 + 4 ppm). K2Cr2O7 stress proved more toxic than CrCl3 on Solanum tuberosum In short, the detrimental effect of high doses of K2Cr2O7 or CrCl3 on Solanum tuberosum can be reduced with suitable combination of plant growth regulators