Agricultural productivity and the quality of crops have been Improved since the time man started horticulture. Today with plant genetics as the scientific tool, improvement in plant breeding is being accelerated. The resulting technology has allowed farmers to manipulate plants so that their propagation with the qualities as desired can be done without waiting for nature to take its course.
Human intervention in plant breeding has taken various forms such as selection, hybridization, and artificial mutation. Artificial mutation is induced either through chemical means or through X-ray and gamma ray radiation. Among the more successful chemical mutagens being used in plant breeding is colchicine, an alkaloid derived from the autumn crocus, Cotchicum autumnale
(Poincelot, 1980). With colchicine treatment, plants may double their chromosome numbers, as oftentimes expressed in the doubling of fruit size or the enhancement of any desired plant quality. Colchicine has been an effective chemical mutagen for a variety of plants. USDA scientists were able to make the small disease resistant Loretto grape produce berries up to three times as large, and bunches two and a half times as large as the normal plant (Science News letter, 1955).
In this present study, there is an attempt to use colchicine treatment in improving the quality of tomato fruits (Lycopersicon). The study seeks to find the effects of colchicine on potted tomato plants. Specifically, the researchers would like to answer the following questions:
1. What are the effects of the different concentrations of colchicine solution on:
a. height of tomato plants
b. flowering and fruiting time
c. weight of fruits
d. cell size of leaves
e. Vitamin C and protein contents of tomato plants?
2. What are the effects of varying time exposure to the different colchicine concentration on:
a. height of tomato plants
b. flowering and fruiting time
c. weight of fruits
d. cell size of leaves
e. Vitamin C and protein contents of tomato plants?
One of the primary considerations that go into having a good crop yield is the use of seeds from plants of high yielding varieties with other desirable traits. Towards this end, genetic researchers have done a tremendous job of improving the yield, and quality of farm products. Among the methods being used are hybridization and mutation through chemicals. X-ray irradiation, and sonar exposure. Some researchers have grown and propagated plants in test tubes so that the genetic make-up of the plants will not be altered through the effects of extraneous materials. Other investigators have met success in producing a extraneous materials. Other investigators have met success in producing a variety of triploid watermelons whose fruits are larger and seedless with the use of colchicine.
This present study which will investigate the effects of varying concentrations of colchicine solution on tomatoes is a parallel study in that it hopes to improve a chosen variety of tomato in height, food value, number of fruits per season, weight of fruits and fruiting time.
The study is concerned mainly with the effects of the different concentrations of colchicine and the varying lengths of exposure to the solution on the phenotypes of TM variety of tomatoes. Only the F of the potted tomato plants were considered. There were forty two (42) treatments with five (5) replicates for each one.
Review of Literature
Davidson, Pertens and Zhao (1983) cited the findings of Eigsti and Dustin on the response of plants and animals to colchicine. The results showed that when proliferating cells were treated for short periods, e.g. 1-3 hours with colchicine, two, (2) responses were observed;
1. Inhibition of spindle formation and arrest of cell development at metaphase stage with chromosomes undergoing increased contraction and disorderly arranged (co-metaphase).
2. Reversion of C-metaphase to the interphase condition. It was also observed that as the cells began to recover from the colchicine treat ment, spindle fibers were formed in the mitotic cells and chromatids segregated at anaphase. However, some chromatids would lag, and the spindle could have three or more poles resulting in a multinucleate cell.
Tetraploid plants may be produced using colchicine. Artificially induced tetraploids usually have larger and thicker leaves and organs, slower and coarser growth, larger cell and pollen grain size, and often reduced fertility (Jules Janick, 1972).
Reese (1951) found out that Avena and Helianthus which were treated with colchicine showed no growth-promoting properties. High concentrations of colchicine instead inhibited hypocotyle elongation. Low concentrations on the other hand, caused slight stimulation.
Ghosh (1950) studied the effects of colchicine on rice. He observed that when sprouted rice seeds were treated with increasing concentrations of colchicine, there was swelling of both radicle and plumule. Treated unsprouted rice seeds had only the swollen plumules. The size of the stomata was not affected by colchicine treatment but the pollen grain size increased. There was retardation in the flowering of plants treated with colchicine. However, treated rice plants gave increased grain yields as compared to the control plants.
A study of the effect of colchicine on Trifolium hybridium was done by Armstrong Robertson (1960). It was found out that the tetraploid plants showed some improvements over the diploid plants in several aspects. In addition, they also observed that the increase in height, leafiness and stem thickness were responsible for the greater hay yield in the tetraploids. Bali and Tandom of India (1959) reported that in Iberes umbellata, colchicine generally increased the size of the stamens, ovary, ovules and pollen. They also noted that pollen fertility and fruit and seed setting in tetraploids were poor though these fruits and seeds were larger than those of the diploid.
The study of Sanders and Franzke (1962) revealed that chromosomes could undergo a reduction in somatic cells following colchicine treatment. This observation supported the idea that somatic cell reduction is one of the mechanisms which gives rise to colchicine — increased true-breeding diploid mutants in certain lines of sorghum.
Armstrong and Robertson (1960) did a chemical analysis of (Trifolium hybridium and found out that the nectar of the tetraploid Trifolium had a slightly higher concentration of sugar than the diploid. However, the diploid was slightly higher in protein and ,ash contents than the tetraploids. Moreover, the Jiploid had lower nitrogen-free extract than the tetraploid.
Screenivasan and Wandrekan (1950) reported that plants treated with Review of Literature
colchicine showed decreased ascorbic acid formation in the earlier stages of germination. These previous studies point out both the beneficial as well as bad effects of colchicine on different plants.
Methodology
The study was conducted on the roof garden of the Ateneo de Davao University. The 210 potted tomato plants were arranged according to the split plot design treatment. All the plants received the required light exposure for tomato crops.
A. Treatment of Tomato Seeds
Viable tomato seeds (TM1 variety) were immersed in different concentrations of colchicine at varying lengths of time.
These were the treatments used in its study:
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B. Analysis of Soil Used in the Study
The soil used in this investigation was obtained from Rapnaga, Ulas, Davao City. It was analyzed by the Bureau of Soils. Recommendations of the Bureau of Soils were followed strictly.
C. Germination of Treated Tomato Seeds
The treated tomato seeds were germinated in IMo. 13 clay pots which were labeled accordingly. The pots which contained 10 seeds each were placed on the roof garden of ADDU. Upon germination, 9 of 10 seedlings were randomly uprooted, leaving only one plant per pot.
D. Measurement of Cell Size
Inprints on the lower epidermis of the youngest leaf of each tomato plant were made using colorless nail polish. These imprints were carefully placed on slides and covered with cover slips. Measurement of the sizes of the cells was done by using the micrometer under high power magnification. The cells measured for the different treatments were chosen at random.
E. Other Parameters Used
Beside size of leaf cells of the tomato plants, other parameters were used in the study:
1. Flowering and fruiting time
The onset of the flowering and fruiting periods for each tomato plant was noted.
2. Height of the plants
Measurement of the height of the tomato plants was done using a meter stick. These measurements were done at thirty-seven (37) days, seventy (70) days and one hundred forty-four (144) days after germination.
3. Weight of the tomato fruits
Ripe fruits were harvested and immediately weighed using the platform balance.
4. Vitamin C content of the tomato fruits
Immediately after harvesting, randomly chosen fruits were air dried and powered for Vitamin C content analysis using the p-dichlorophenolindphenil method (Pearson, 1970).
5. Protein content analysis
Two grams of the powdered tomato fruits from each treatment was analyzed for protein content using the macroKjeldahl method (Osborne, 1978).
Discussion and Analysis of Results
In this study, several parameters were used to find out the effects on the tomato plants of the different concentrations of colchicine and varying lengths of exposure of the seeds to the colchicine solutions. These parameters are the height of plants, the onset of the plant’s flowering and fruiting, weight of the fruits, cell size of leaves, and Vitamin C and protein contents of the fruits.
Plant Height
Tables 1,11,11a, b, and c show that increasing concentrations of colchicine and increasing lengths of exposure time produced significant differences in the height of the control and experimental plants during the early vegetative days but produced no significant differences at maturity. Treated plants were generally shorter at the start but were able to outgrow untreated plants as they approached maturity. The tallest plants were those grown from seeds exposed to 0.50 to 0.75 percent colchicine. Increasing concentration higher than 0.75 percent tended to decrease height. Plants exposed to colchicine for 6 hours were generally taller, and increasing exposure time longer than 6 hours tended to decrease height. However, no consistent pattern of response relative to increasing exposure time was observed.
At 37 days from sowing, the plants treated with colchicine were generally shorter than the control which had a height 8.43 cm. Increasing colchicine concentration depressed height and a drastic reduction became very apparent at concentrations higher than 0.75 percent. The shortest plants with mean heights of 5.67 cm. were from seeds exposed to the highest colchicine concentration of 2.0 percent. Increasing exposure time produced significant but inconsistent
differences on plant height within a range of 6.0 to 8.75 cm. At this stage, plants exposed to colchicine for more than one hour were generally taller than those exposed only for one hour.
Table I. Influence of Varying Lengths of Exposure of Tomato Seeds to Different Concentrations of
Colchicine on Height of Tomato Plants.
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Table IIa. ANOVA Results on the Influence of Varying Lengths of Exposure of Tomato Seeds to Different Concentrations of Colchicine on Plant Height 37 days after Sowing.
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Table 11b. ANOVA Results on the Influence of Varying Lengths of Exposure of Tomato Seeds to Different Concentrations of Colchicine on Plant Height 70 Days after Sowing.
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Table 11c. ANOVA Results on the Influence of Varying Lengths of Exposure of Tomato Seeds to Different Concentrations of Colchicine on Plant Height 144 Days after Sowing.
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At 70 days of age, all treated plants were taller than the 58.77 cm. of the control. Maximum height was at 0.25 percent colchicine at 69.0 cm. The retarding Influence of Increasing concentrations on plant height continued to be exhibited at concentrations higher than 0.25 percent. Inconsistent response to increasing exposure time continued to be exhibited Inspite of the resulting significant differences In height (Table lib). Plants exposed to colchicine for 6
hours or more also came out taller than those exposed for only one hour.
At maturity (144 days) all colchicine treated plants were taller than untreated ones by about 5 to 11 cm. Maximum height of 89.69 cm. was observed in the plants treated with 0.50 percent colchicine. However, statistical analysis showed no significant differences (Table He). Similarly, no significant differences in height were observed with increasing exposure time inspite of the tendency of plants exposed for 6 hours or more to be taller than those exposed for only one hour. Statistical analysis showed no significant interaction between colchicine concentration and exposure time.
Yield and Yield Components of Tomato
Yield components of the tomato plants include the number of days from sowing to flowering and the number of days from sowing to fruiting. Tables III and IVa & b show the data on the yield components of the tomatoes. Tables IVa and b reveal that increasing concentrations of colchicine significantly delayed the onset of flowering but produced no significant differences in the fruiting time. Increasing exposure time had no influence on the length of time from planting to flowering and fruiting.
The earliest flowers produced in 48.33 days were in the untreated plants as shown in Table III. Exposures to increasing colchicine concentrations resulted in an increasing delay in the flowering (55.29 days) to a maximum of about 7 days in 2.0 percent colchicine.
As for the fruiting time, although the tendency of increasing colchicine concentration to delay continued to be exhibited, no significant differences were observed (Table IVb) between the control and the treated plants.
Table III. Influence of Varying Lengths of Exposure of Tomato Seeds to Different Colchicine Concentrations on Number of Days from Sowing to Flowering and Number of Days from Sowing to Fruiting.
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Table IVa. ANOVA Results on the Influence of Varying Lengths of Exposure of Tomato Seeds to Different Concentrations of Colchicine on Number of Days from Sowing to Flowering.
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Table IVb. ANOVA Results on the Influence of Varying Lengths of Exposure of Tomato Seeds to Different Concentrations of Colchicine on Number of Days from Sowing to Fruiting.
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Fruit Yielding
Table VI shows that increasing concentrations of colchicine and length of exposure time significantly increased the yield of tomato to a maximum point beyond which yield a decline was observed. Optimum yields were observed in plants from seeds exposed for 24 to 48 hours in 0.75 to 1.5 percent colchicine concentrations (Table V).
From 399.62 gms. of yield in the untreated plants, treating seeds with colchicine concentrations of 0.25 to 1.5 percent increased the yield to 402.06 and 418.90 gms. respectively or 4.8 percent higher than that of the control. Optimum yields were in colchicine concentrations of 0.75, 1.00 and 1.50 percent with yields of 415.14,417.40 and 418.90 gms. respectively. Plants exposed
to 2.0 percent concentration yielded only 287.05 gms. which is significantly lower than the control.
Increasing exposure time resulted in increasing yield with the optimum at 24 and 48 hours which registered yields of 462.65 and 452.15 respectively. Compared to 293.58 gms. yield from plants exposed for only one hour, this represented an increase of 57.6 percent Exposing seeds for 72 hours resulted in declining plant yield to 356.22 grams.
Interaction between colchicine concentration and length of exposure time was significant indicating a relationship where the effect of increasing exposure time is enhanced with increasing concentration.
Table V. Influence of Varying Lengths of Exposure of Tomato Seeds to Different Colchicine Concentrations on Fruit Yield.
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Table VI. ANOVA Results on the Influence of Varying Length of Exposure of Tomato Seeds to Different Concetrations of Colchicine on Total Fruit Yield.
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Cell Size
Table VII reveals that increasing the concentration of colchicine from 0 to 0.50 percent also increased the size of the cells in the leaves of the tomatoes. Maximum size of 10.32 microns was reached with 0.50 percent concentration. Further increase in the concentration reduced the size of the cell until it reached the smallest size of 8.26 microns at 2.00 percent concentration. These differences in cell size were significant. (Table VIII).
On the other hand, the biggest cell measuring 8.81 microns came from tomatoes exposed for only one hour. Increasing the exposure time tended to decrease the size of the cells. The decrease, however, was not consistent although it was significant. The smallest cell, measuring 7.36 microns was obtained from tomatoes exposed to colchicine for 12 hours. The effect of concentration on the cell size varied with exposure time. The interaction effect was significant.
Table VII. Influence of Varying Lengths of Exposure of Tomato Seeds To Different Colchicine Concentration on Cell Size.
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Table VIII. AIMOVA Results on the Influence of Varying Lengths of Exposure of Tomato Seeds to Different Concentrations of Colchicine on Cell Size.
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Vitamin C Content
Table IX shows that increasing concentration of colchicine increased the vitamin C content of tomatoes, except at 0.50 to 1.00 percent where the vitamin C content decreased. The highest Vitamin C content was attained at 2.00 percent colchicine. These results indicate that colchicine treatment of seeds can be used to enhance the Vitamin C content of the plant (Table X).
Varying lengths of exposure time also resulted to significant differences in the Vitamin C content of tomatoes. No consistent pattern of increase or decrease, however, is evident. Vitamin C content was at its highest in the plants grown from the seeds with 24 hours exposure while the lowest was noted at 12 hours of exposure.
Interaction between concentration and exposure time was highly significant. This means that the effect of different levels of concentration on the Vitamin C content varied with different lengths of exposure to colchicine.
Table IX. Influence of Varying Lengths of Exposure of Tomato Seeds to Different Colchicine Concentrations on Vitamin C content.
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Table X. ANOVA Results on the Influence of Varying Lengths of Exposure of Tomato Seeds to Different Concentrations of Colchicine on Vitamin C content
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Protein Content
Table XI reveals that as the concentration of colchicine increased from 0 to 0.75 percent, the protein content decreased from 0.97 to 0.83 mg/IOOg. Further increase in concentration, however, resulted to arr increase in the protein content. Optimum amount of protein was obtained at 1.50 percent concentration. The differences in protein content due to the varying concentrations of colchicine were highly significant (Table XII).
The effect of exposure time on the protein content of tomatoes was not consistent just like that of the effect of concentration of colchicine (Table XI). As the exposure time increased from 1 to 24 hours, the protein content increased from 0.93 to the optimum level colchicine (Table XI). As the exposure time increased from 1 to 24 hours, the protein content increased from 0.93
to the optimum level of 0.99. Further exposure time seemed to decrease the protein content. The effect of exposure time on the protein content of tomatoes was also significant (Table XII).
The AIMOVA also revealed a highly significant interaction effect of concentration and exposure time.
Table XI. Influence of Varying Lengths of Exposure of Tomato Seeds to Different Concentrations of Colchicine on the Protein Content.
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Table XII. ANOVA Results on the Influence of Varying Lengths of Exposure of Tomato Seeds to Different Concentrations of Colchicine on Protein Content.
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Summary, Conclusion and Recommendations
This study attempted to find the effects of colchicine on the phenotypes of potted tomato plants. Specifically, the researchers wanted to know.the effects of the different concentrations of colchicine solution and of varying time exposure of the seeds to the different colchicine concentrations on tomato plants
a. height
b. flowering and fruiting time
c. weight of fruits
d. cell size of leaves
e. Vitamin C and protein contents
The study showed that increasing colchicine concentrations and increasing lengths of exposure time produced significant differences in the height of the control and the experimental plants during the vegetative days. There was no such significant difference, however, in the height of the control and the experimental plants at maturity.
The increasing concentrations of colchicine significantly delayed the flowering time of the tomato plants. There was, nevertheless, no significant difference in the fruiting time of the control and the experimental tomato plants.
The study, likewise, revealed that increasing concentrations of colchicine and length of exposure time increased the yield of tomato significantly. Maximum yield was observed in the plants grown from seeds exposed for not more than 24 hours to not more than 1.5 percent colchicine concentration.
The largest cell (10.32 microns) was observed in the 0.50 percent colchicine concentration. Beyond the said concentration, size of the cells decreased. The differences in cell sizes were found to be statistically significant. On the other hand, the biggest cell was observed in plants from seeds exposed to only one hour in colchicine solution. Vitamin C content increased significantly as colchicine concentration, as well as the length of exposure time increased. The highest Vitamin C content was observed in plants coming from seeds exposed for 24 hours to colchicine.
This study also showed that protein content in the tomatoes was at its highest at 1.5 percent concentration. The differences in protein content due to varying concentrations of coichicine were found to be highly significant. Furthermore, the highest protein content was observed in plants exposed to coichicine solutions for 24 hours. Statistical test also showed that the differences in protein content due to varying lengths of exposure time was highly significant.
In conclusion, this study has positively indicated some effects of coichicine on the growth and development of Lycopersicon esculentum. The effects of the alkaloid mutagen varied with its concentration, the length of exposure of the seeds, and the developmental stages of the plant. Thus, while increasing concentrations produced significant differences in the heights of the control and experimental plants during their vegetative growth, no such effect could be observed at maturity. Likewise, while higher concentrations and longer exposures significantly increased the yield and the Vitamin C and protein contents of the fruits, these delayed the flowering time of the plants. High concentrations also reduced cell size. This latter observation seems to support the findings of Sanders and Franzke (1962) which revealed reduction in somatic cells following coichicine treatment.
Recommendations:
The researchers would like to recommend the following points to future investigators:
1. a similar study can be done using tomatoes in garden plots.
2. Chromosomal analysis of the tomato cells from different parts of the plant be made.
3. the extension of the study of the F2, F3 and F4 generations to determine if the characteristics induced in the parent plants by varying colchicine concentrations and varying lengths of exposure time, are inherited.