Mutation breeding of in vitro grown Begonia rex: morphological variations and mutagenic effects

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.

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)

It is reported that in the leaves of Begonia "the portions on either side of the midrib are usually quite unlike; and there are marked internal asymmetries, as when two daughter cells are dissimilar. In most cases of this sort, however, the asymmetry proves to be part of a larger and more complex pattern which is symmetrical. Symmetry is often more conspicuous in embryonic structures or meristematic regions than at maturity, and some of its most remarkable examples. Neel (1940) reports that in Begonia where there are "...silver spots on the leaf surface, there is a hair formed in each spot save the very small ones, and the larger the spot, the longer the hair." (cited in: 3rd Symposium on Haploidy, 1984)

It is reported that root and shoot restoration on leaves which have been removed from the plant and in which petiole has been placed in the soil has occurred and that in such cases "...after adventive roots and buds have been formed on lamina or petiole, various anatomical changes may be observed, especially a marked increase in the vascular tissue of the petiole. Furthermore, instead of being disposed in an arc, as in normal petioles, this tissue often enlarges to form a complete vascular ring. The petiole thus becomes structurally as well as functionally a stem. Simon (1929) is stated to have noted this in the Begonia and stated that the "...bundles from the young roots induced new vascular tissue only in that part of the petiole just below them, suggesting the polar distribution of a hormone. Similar results were obtained by Doyle (1915) through grafting buds onto the petioles of rooted leaves." (3rd Symposium on Haploidy, 1984)

The location of the regenerated structures is determined mainly by the anatomy of the leaf." However, it is important to note that in some species, including that of Begonia rex that while not always succulents "...restoration and vegetative reproduction occur in other parts of the leaf than the base." (3rd Symposium on Haploidy, 1984) In Begonia rex it is stated that "...shoots may be induced very readily on petiole and blade by removing the growing points of the shoot. Prevot (1938, 1939) found that these arose from epidermal cells, but only after they had reached a certain stage of maturity. Hartsema (1926) has described the changes that such cells undergo at the start of shoot development (strong protoplasmic streaming, migration of the nucleus, and increase in cytoplasm).

Here and in a good many other plants, shoots will appear on the blade if one or more of the veins are cut thus perhaps preventing the access of inhibiting substances. Prevot was able to induce bud formation on begonia leaves by the application of various substances and by growing the plants in the absence of oxygen. He also found that strips of epidermis removed from the leaf would form buds. Not all begonias have high regenerative ability, and this seems to be an inherited character when various types are crossed." (3rd Symposium on Haploidy, 1984) Begonias are generally reproduced in cultivation by plantlets and their formation on leaves being removed and then located in an atmosphere that is humid. From individual cells of the upper epidermis shoots develop, and roots originate from parenchymatous cells near the boundary between xylem and phloem in the veins." (3rd Symposium on Haploidy, 1984)

The work of Janick (1997) entitled "Plant Breeding Reviews" states that plant tissue culture techniques "are suited to the dissociation of plant chimeras. When mutation breeding programs yield complex mosaics and patches of genetically unique cells appear on organs, tissue culture can be used to generate fully mutant plants. In Begonia for example sectored chimeras appear following whole-plant irradiation and in vitro organogenesis was used to recover nonchimeral mutants from mutant sectors. (Shigemasu and Matsubara, 1972, in: Janick, 1997, p. 67)