Mutation Breeding of in Vitro

Mutation Breeding of in Vitro Grown Begonia Rex and Analysis of Morphological Data

The purpose of this study is to examine: (1) the wide spectrum of morphological variations induced by mutation breeding; (2) the effect of mutagenic agents (chemical mutagens) such as colchicine on mutation frequency; (3) the effect of the physiological state of explants material on mutation frequency; (4) the effect of chemical mutagen dosage (colchicine) on mutation frequency; (5) the effect of mutation breeding on leaves for ornamental foliage plants; (6) mutation on leaf number and morphology using colchicine as chemical mutagen; (7) the effect of mutation breeding on shoots number using colchicine as chemical mutagen; and (8) the effect of mutagen (colchicine) on change leaf color in begonia rex. Toward this end, this work will conduct a review of literature relevant to mutation breeding of in vitro grown begonia rex and conduct an analysis of the morphological data.

Introduction

The target of the in vitro grown plant method in the ornamental nursery industry is changing morphological characters and the most important morphological traits that are targeted by breeders across the world are the flower color, size of the flower and the color of the leaves of the flower. (Borertjes and Harten, 1978)

III. The wide spectrum of morphological variations induced by mutation breeding:

Studies show that improvement to the morphological trait can occur through plant breeding including the plant yield, the plant toleration to stress in the environment and to any morphological character. There are two classes of morphological traits as follows:

(1) Quantitative; and (2) Qualitative.

Examples of quantitative morphological traits are such as:

(1) Plant productivity;

(2) Number of fruits;

(3) Seeds; and (4) Leaves.

Examples of qualitative morphological traits are such as:

(1) Sterility;

(2) Fertility; and (3) Leaf Color.

Three essentials factors are necessary to consider in the process of inducing mutation in a plant and those are stated as follows:

(1) Plant variety;

(2) Mutagenic agent;

(3) Dosage of mutagenic agent;

(4) Physiological state of explants material.

IV. Effect of Different Factors on Mutation Frequency

Results that have been obtained in different experiments show that different mutagenic agents produce different frequencies in differing plants therefore; the determination of the optimum mutagenic agent for inducing mutation is a primary factor in mutation breeding experiments. The work of Rao (1984) demonstrated that pre-soaking seeds treated with Sodium azide (NaN3) resulted in the creation of high mutation frequency in Oryza sativa when compared with other chemical mutagens.

In comparison, combining EMS and gamma rays resulted in the production of more chloroplast mutation frequency than did sodium azide treatment according to the study reported by Bokhari and Ahmed (1990). Therefore, it can be understood that the type of mutagenic agent is a strong factor in determining the mutation frequency.

V. Effect of plant variety on mutation frequency

Various experiments involving application of mutagenic agents on plants indicate that differing cultivars for the same crop provide different responses to treatment with mutagenic agents in relation to their susceptibility to the mutagenic agent. There is less susceptibility to Vigna radiate cv. G65 than to cv.PS16 to EMS, Hydrogen azide and gamma rays according to the work reported by Khan (1989). In fact, differing varieties of Arabidopsis thaliana when treated with EMS results in the plants generated varying significantly in fatty acid profiles. The variety 9al showed mutation that inhibits the production of linolenic acid production and exhibited the production of paltmitin acid. However, cv. 1A9 showed a decrease in linolenic acid and an increase in oleic acide compared to 2a11 that showed a decreased in linolenic acid and an increase in steric acid. Based on these findings it is indicated that differing plants show different responses to mutagenic agents.

VI. Effect of the physiological state of explants material on mutation frequency

Various types of explants show differing responses to mutagenic agents depending upon their physiological state. The work of Singh et al. (1989) demonstrated that the explants with more active growing cells result in the production of more mutation. Singh et al. reports that indications are that seeds that are pre-soaked are more sensitive to EMS than are dry seeds, because soaking the seeds breaks the seed dormancy and begins germination and this leads to a higher mutation frequency.

VII. Effect of dosage on mutation frequency

The work of Ventakeswaralu, et al. (1988) demonstrated that an essential factor in mutation frequency is the dosage of the mutagenic agent. This arises from the concept that mutation can be inducted by the mutagenic agent up to a specific concentration however; any dosage over that concentration increases mortality as the dosage then becomes lethal in nature. The result is difficulty in determining the optimum mutagen dosage for each specific plant. Different mutagenic agents given in differing dosage resulted in different efficiencies in mutation and in differing effectiveness of mutation. These terms may be defined as follows:

(1) Mutation frequency = rate of mutation / lethality

(2) Effectiveness = rate of mutation / dosage * duration

(3) Factor of effectiveness = number of mutations / number of treated explants *100

VIII. Survival rate vs. optimum mutation frequency

The frequency of mutation increases when the dosage of radiation is increased or when the concentration of the chemical mutagen is increased however, there is a known decrease in the survival rate. The primary objective of mutation breeding is to gain a balance between the rate of mutation and the rate of survival. Furthermore, in mutation breeding experiments, it is critical it know the mutagenic agents' impact on plant mortality prior to the use of the mutagenic agent. Calculation of the ration of mutation frequency and survival is required in order to determine the optimum concentration. The relationship between the dosage, mutation frequency and survival was studied and reported in the work of Venkateswaralu who states findings that the rate of survival is inversely proportional to the dosage of mutagenic agents, and the mutation frequency is directly proportional to the dosage of the mutagenic agent.

IX. The Effect of Mutation Breeding on Leaves

The ornamental foliage industry is a great supporter of many country economies and an essential part of this industry is that of plants with new characters in their leaves. The primary characteristics of plants leaves are those of:

(1) Color;

(2) Size;

(3) Shape; and (4) Number of leaves.

Mutagenic agents have the potential to change the characteristics of plant leaves and to produce new varieties of foliage. For example, the work of George, Hall and De Klerk (2007) entitled: "Plant Propagation by Tissue Culture" reports that the inhibition of direct root and shoot formation on isolated leaves of Begonia rex was accomplished by use of "I-10mg/1 GA3 being overcome by adding 2.4-D at an equivalent concentration." (p.231)

The work of Doorhenhos and Karper (1975) and Mikkelsen and Sink (1978) demonstrated that in Begonias adventitious shoots may arise from single epidermal cells. It is reported that there are primary advantages to having both NO3 and NH1 ions in the medium in that uptake of one results in a better pH environment for the uptake of the other and the result is stabilization of the medium.

The medium of De Jong et al. (1974) is stated to have "always had a pH of 4 9-5.0 after Begonia buds had been cultured." (George, Hall and De Klerk, 2007) It is reported that the majority of plant cells and tissues in vitro are able to tolerate pH in the range of approximately 4.0-7.2. (George, Hall and De Klerk, 2007, paraphrased)

Heide (1968) states findings that shoot bud formation on isolated Begonia leaves were enhanced "when the leaves were treated with abscisic acid, and inhibited when either auxin or gibberellin was applied. Seasonal variation in the capacity of Begonia leaves to produce shoot buds was thought to be associated with variation in endogenous ABA levels." (George, Hall and De Klerk, 2007) The work of Shepard (1980) reports findings that adding ABA to the growth medium caused morphogenesis to occur more rapidly." (George, Hall and De Klerk, 2007)

X. Mutants with Different Leaf Number and Morphology

The work of Nair and Abraham (1989) states that in the ornamental plant industry that it is important economically to improve the photosynthetic capacity to increase the number of leaves and to increase the plant yield. It is reported that Yam bean when treated with EMS and Gamma radiation resulted in a higher number of leaves being produced.

Mutations with various leaf size are popular in the breeding of mutation in plants. The work of Gottshallk and Wolf (1983) states that EMS treated with Vigna radiate created high leaf area. Jugran et al. (1985) report the production of long and narrow leaves being induced in Cajanus cajan through use of 0.2% EMS and report as well the reduction in leaf size commonly occurred with gamma irradiated winged bean. These variations were common with farmers due to high yield.

Mutation modifies the variation in the leaf shape. The change in the size of petiole is common in some mutants. The size of the petiole can be increased or decreased through use of gamma radiation.

XI. The Effect of Mutation Breeding on Shoots and Stem

The initiation of shoots is controlled by the large number of genes in higher plants. This is clearly demonstrated in the alternations in shoots resulting following treatment with a mutagen. The increase in the number of shoots has many benefits which includes the increase in the number of branches which can ultimately result in the increase of yield according to Savov (1983). Furthermore, the proportion of shoots to the length of internodes is an essential factor of plants and in Malus pumila treated with gamma radiation demonstrated is an improvement in shoots and internodes ration. Therefore, the growth of the plant resulted through an increase in the number of branches. (Paprstein, 1988)

XII. The Effect of Mutation Breeding on Plant Height

A change in the height of a plant is an important horticultural characteristic however, plant height in mutation breeding is not a common feature as tallness in a plant is not a desired characteristic since an increase in the height of a plant results in a negative impact on the plant stem stability.

XIII. Additional Morphological Information

The work of Arora, Nakao and Nakajima (1970) entitled "Perpetuation of Begonia Rex by Aseptic Culture with Micro-Leaf Cuttings Under Various Conditions of Auxin and Cytokinin" state that they conducted a study in which micro-leaf cutting of Begonia rex were aseptically cultured with the aim to isolate and propagate the irradiation induced chimeras." (p.275) Micro-leaf cutting of Begonia rex are stated to have regenerated roots and a few buds when they were cultured ascetically on White's basal medium. It is stated that the lower concentrations of NAA "...showed stimulatory effect on root initiation which was observed 35 days following inoculation. Callus tissue was formed by the varying concentration of NAA microcultures and the maximum number of roots per rooted culture is stated to have been "recorded at 0.1ppm of NAA." (Arora, Nakao and Nakajima, 1970, p.276)

It is however reported that the lowest concentration of NAA as well as in control where callus did not form at all, in a few cultures a few buds were formed whereas as relatively higher concentrations bud formation was completely suppressed. No root formation took place during the thirty day period when the temperature maintained was between 17~22 degrees Celsius however following one month of the temperature being increased to 27~30 degrees Celsius most of the culture began regeneration of roots.

It is reported that the combination with the 0.1 ppm NAA that the maximum percentage of culture regenerated roots on the 25th day however in the combination with 9.91ppm NAA organ differentiation was constant until the 35th day however there was a sudden increased noted up until the 50th day.

Arora, Nakao and Nakajima state that a "number of species in the genus Begonia are well-known for their ability to form both adventitious buds and roots on detached leaves without the aid of plant regulators." (1970, p.280) It is reported that Heide (1965) reported that the "stimulatory effects of these substances in the asceptic culture of Begonia with expanding leaves of about 3.5 cm diameter along with petioles trimmed to a length of 2cm and it is stated that the results of the experiment reported in the work of Arora, Nakao and Nakajima provide explanation that in the asceptic propagation of Begonia with micro-leaf cuttings of 3X3 mm auxin and cytokine are needed to produce speedy and better generation of organs." (Arora, Nakao and Nakajima, 1970, p.280)

In fact, it has been reported in previous studies including those of Heide (1965) and Wirth (1960) that B. rex requires a high cytokinin to auxin ratio for bud formation to be stimulated and root formation to be suppressed. A low ratio of cytokinin to auxin ratio has the opposite effect in that root suppression does not occur and bud formation is not stimulated. NAA at a high concentration further promoted the development of roots and inhibited the formation of buds while the high concentration of kinetic in turn promoted formation of bud to the maximum degree and almost but not quite inhibited root formation. It is however reported that the same combination with auxin and cytokinin in equal concentration and the difference in the percentage of roots and buds of cultures can be credited to the developmental stage of the mother plants and the leaves' age due to the fact that during collection of leaves it was not possible to precisely determine the ages of the leaves. NAA being applied following kinetic resulted in the production of better differentiation of organs than the two combined. Buds were regenerated by cultures on kinetin media alone at a low range of temperature (17~22 degrees Celsius) however the majority of cultures took one month for regeneration of buds and only when the temperature range was increased to 27~30 degrees Celsius with culture also regenerating roots.

It was further noted that Heide (1965) reported "in Begonia that high temperature suppresses bud formation and counteracts the promotive effect of cytokinins on this process and the inhibitory effect of cytokinin on root formation." (Arora, Nakao and Nakajima, 1970, p.281) Therefore it is stated to be evident that "for early differentiation of organs with better growth in the multiplication of B. rex by aseptic culture with micro-leaf cuttings, auxin and cytokinin are required in a defined ration or separately at certain stage of organ development under favorable temperature conditions." (p. 282)

High success is possible to achieve when "micro cuttings are first cultured on kinetin medium and later transferred to NAA medium where almost all of the cultures regenerated roots as well as buds with better growth." (Arora, Nakao and Nakajima, 1970, p.282) The most effective for speedy regeneration of organs is stated to be a combination of NAA (0.01ppm) and kinetin (0.1ppm)." (Arora, Nakao and Nakajima, 1970, p.283) In addition, this method may be applied with the benefits of isolation and diversion of irradiation induced chimeric tissues into fully homogenous plants." (Arora, Nakao and Nakajima, 1970, p.284)

The article "Genetic Manipulation In Crops: Proceedings of the International Symposium on Genetic Manipulation in Crops, the 3rd International Symposium on Haploidy, the 1st International Symposium on Somatic Cell Genetics in Crops, Beijing, October 1984" reports that the use of colchicine in culturing the explants of Begonia rex were successful when propagated in vitro. Furthermore, "the plantlets of Begonia rex...with fancy leaves cultures in vitro remained true to their original types." (1984) It is reported that Lindemuth (1904) "removed and rooted mature begonia leaves and observed that they then increased considerably in size, chiefly because of cell enlargement. While they were attached to the plant this presumably was prevented by "correlative inhibition." Similar results have been reported by others." (3rd Symposium on Haploidy, 1984)