F. Majd, E. Jahangirzadeh, S. Vedadi, M. Naseri Tafti, J. Rastegari Nuclear Research Center for Agriculture and Medicine,
Karaj, Iran
Abstract. Genetic variation is an essential component of crop breeding. Induced mutations are highly effective in enhancing natural variability of genetic resources, and have been instrumental in developing improved cultivars of crops including fruits crops. Recent advances in biotechnological techniques have shown a great potential as efficient methodologies for vegetative micropropagation, screening techniques and genetic characterization including mutation induction. On this basis, a local, well- adapted and widely-consumed Clementine cultivar was selected for introduction into a mutation induction programme in order to reduce the excessive seeds in the fruits. A physical mutagen (γ-ray) was applied at doses of 35, 40 and 45 Gy on selected seedling apical buds. Irradiated buds were grafted onto sour orange root stocks (M1V1) and chimeras disassociated by further vegetative propagation. Finally M1V3 plants were transferred to the field and after the production of fruits, selection for the desired fruit types was undertaken. The results showed that the radiation treatment was able to produce mutant genotypes with seedless fruits, early and late ripening and cold tolerance.
1. Introduction
Since 1930, about 130 citrus cultivars of oranges, tangerines, sweet lemons, sour lemons, grapefruits and other hybrid lines have been introduced in Iran. Out of these introductions, and after preliminary cultivar comparisons in different research experiment stations, a number of selected citrus cultivars have been distributed in three regions of the country including the Caspian Sea region (Gillian & Mazandaran), the central region (Kerman, Fars, Kermanshah, Ilam, Kohkilouyieh, Bouyer Ahmad and Khorasan provinces) and finally along the Persian Gulf and Oman Sea (Khoozestan, Boushehr, Hormozgan and Sistan Balouchestan provinces).
The total area of citrus fruits cultivated amounts to 235000 hectares, of which 92% is productive and 8% is non-productive (juvenile trees). Mazandaran province ranks first in the list with 37%-of the total cultivated area. Fars, Jiroft, Kahnooj, Hormozgan and Kerman areas respectively have 20.7, 14.8, 13.2 and 5.2% of the area. The rest, which is about 8.7%, is spread into other provinces (Table I).
2. National production
Tangerine ranks third in total citrus production in Iran. Tangerine varieties cultivated in Iran include, unchiu, younesi, page, oneco, kino and clementine. Clementine has been introduced into Iran along with unchiu. It originates from Algeria; and can be cultivated in the Caspian, Jiroft and other regions with a moderate climate. In Table I the cultivated area (productive &
non-productive), production and yield performance data are given. Tangerine potentials for production, especially for export, can be considered to be well above its present status.
Clementine variety is well adapted to both the climate and other biotic and abiotic stresses in the north of Iran and has a constant high productivity. The induction of mutation of one or a few characters through the use of physical mutagens has proved to be an efficient tool for enhancing the frequency of spontaneous mutations [4, 5]. According to a literature review, somatic mutations have contributed to the development of new varieties in many fruit tree species.
TABLE I. CULTIVATED AREAS, PRODUCTION AND YIELD PER HA IN CITRUS FRUIT IN semi-dwarfness) and the initiation of compact types (due to changes in the plant architecture, an increase or decrease in lateral branching or changes in branch position), changes in the flowering and ripening period, an increase in pollen sterility and changes to fruit size, shape or the number of seeds. The aim of this research proposal was to induce seedless/ less seeded Clementine from the presently seeded type through the use of physical mutagenesis.
3. Materials and methods 3.1. Shoot- tip grafting
In vitro grafting of shoot- tips has been widely used to obtain virus-free plants. Citrus plants obtained by this technique do not possess thorns or juvenile characters. Therefore, shoot- tip grafting can be used as a procedure for the production of true – to- type, virus-free, non- juvenile plants.
Seeds of Ponsiruse (which has trifoliate leaves and serves as a morphological marker for the identification of adventitious buds produced by the root stock) were sterilized for 10 min in 0.7% sodium hypochlorite solution and rinsed three times with sterile distilled water. After removing the seed coats, seeds were cultured on Murashige and Skoog medium [2] solidified
with agar. Seeds were incubated for two weeks in continual darkness and at 26 ±1ºC of temperature [3].
Within 8 to 15 days, numerous buds sprouted. Shoots shorter than 3 cm long were then used as source of shoot tips. They were stripped of larger leaves, cut to about 1 cm long, surface-sterilized by immersion in a 0.25% sodium hypochlorite solution containing 0.1% tween-20 wetting agent for a few minutes and then rinsed three times with sterile distilled water.
3.2. Grafting
Two week- old Ponsiruse seedlings were decapitated under aseptic conditions, leaving about 15 mm of each epicotyl. The roots were also cut to a length of 40-60 mm, and the cotyledons removed. A vertical incision, 1mm long, was made in the stem, starting at the point of decapitation, followed by a transversal incision of at the 1-2mm long bottom. These cuts were made through the cortex to the cambium, and the flaps created by these incisions were lifted slightly to expose the cortex. A shoot tip was then placed on the vascular ring in the opened flaps of the incision at the top of the decapitated epicotyl. The grafted plants were subsequently placed on paper bridges over MS liquid medium supplemented with 75g/l sucrose and maintained at a constant temperature of 26±1ºC in a 16hr daily photoperiod of 1000 Lux illumination (Figure 1.A-B,C).
Thirty days after grafting, approximately 80% of the grafted seedling wounds showed advanced recovery. After five to six weeks, the successfully - grafted shoot- tips produced 1 to 4 young leaves, but only 45% of the original number of grafted seedlings survived.
During scion growth a number of buds emerged from the root stock. Taking into consideration that Ponsirus leaves are trifoliate and Clementine leaves are unaffiliated it was therefore possible to expedite scion growth by cutting off adventitious Ponsirus buds like (Figure 1.E, F).
When the root and scion length reached 7 to 8 cm respectively, and primary leaflets showed suitable growth, plantlets were removed from the culture medium and following a thorough rinsed with water, planted into sand filled pots. The experiments were watered every week using MS medium, containing only macro and micro-salts. Three weeks later fully developed plants were transferred into bigger pots containing sand, clay and animal manure in 1:1:2 proportions (Figure 1 F).
3.3. In vitro mutation induction
Bacterial and virus free tangerine scions were irradiated with gamma ray (60C0) using doses of 0, 35, 40, and 45Gy. Irradiated scions were then grafted onto sour orange rootstocks (600 individuals for each dose). At the end of the growing season, M1V1 shoots were cut back at the second basal bud and three to eight buds were then individually re-grafted on separate rootstocks.
To isolate induced mutants exhibiting chlorophyll abnormalities the same procedure was repeated on the original rootstocks with two – bud scions the following year. In M1V2 and M1V3 a number of morphological mutations affecting, leaf size, shape, and reduced internodes length were detected and recorded. M1V3 plants were later on transferred to the field (Figure 1G) and, after fruit production, an evaluation for fruit shape and colour, number of seeds per fruit, cold resistance and earliness was performed.
4. Results and discussion
Analysis of variance indicated that the grafting success in the two hormonal pre-treatment had significant differences at the 1% level (Table II). WE may conclude that an application of 2,4-D as a hormone pre-treatment produced successful grafting in more than 17% of the grafts while, in the case of the BAP pre-treatment, only 8% successful grafts were produced.
Decreasing the sucrose concentration to 7% in the medium compared to 8.5%, 10%, and 11.5% had also significant positive effects on the success rate of grafting. Considering the interaction effect of hormonal pre-treatment and sucrose concentration, it was determined that the 2, 4-D pre-treatment with a 7% sucrose concentration produced the best results.
TABLE II. OVERALL RESULTS OF THE PERCENTAGE OF SUCCESSFUL GRAFTING OF HORMONAL PRE-TREATMENT AND SUCROSE CONCENTRATION, ON THE BASIS OF ANALYSIS OF VARIANCE
Sources of variance Degrees of freedom Mean percentage of successful grafting
** Significant difference at 1% level
M1V3 plant after three years in the field have reached to 80% of productivity. 2000 trees were evaluated in the main field for the following traits: number of seeds in fruits, earliness, late maturity, cold tolerance and other favourable traits.
As shown in Table III and Figures 9 and 10, six (6) mutated seedless trees were obtained, all deriving from a 40Gy of Gamma ray irradiation assay. We also identified 13 cold tolerant trees. This trait is very important for the citrus production areas of the country, since it often happens that the temperature drop below zero. Out of these 13 trees, seven trees were resulted from irradiation with 35 Gy and six from irradiation with 40 Gy Gamma rays dose. There were significant differences in the maturation dates; two trees were early maturing, eight mid maturing and four late maturing trees. These latter traits are likely to be very well accepted by farmers, since this should allow having fresh fruits for a longer period of time and therefore present better marketing opportunities.
TABLE III. ANALYSIS OF FRUIT QUALITY IN THE FIRST YEAR OF PRODUCTION
Dose Early
The Total Soluble Solids (TSS) scale for all selected trees was determined and determined between 9 to10, except for one of the late maturing trees, which had a 6.8 value. The TSS of Clementine varies from 8.5 to 10 in the region. These findings need to be confirmed in the future, prior to release and commercialization.
The seedlessness trait was observed the following year, confirming the stability of the mutation for that specific trait (Figure 2).
Figure 1: Infected citrus plants growing in pots in the green house were completely defoliated by hand and placed in a warm green house, or in a growth chamber.
Figure 2: Seedless tangerines compared to the parental type (A, B, C), late maturing tree (D).
REFERENCES
[1] AGRICULTURE STATISTIC YEAR BOOK OF IRAN Ministry of agriculture Department of budget and planning - Information Statistic unit, Public, (2000) (98-99).
[2] MURASHIGE AND SKOOG MEDIUM A revised medium for rapid growth and bioassays with tobacco tissue culture. Plant Physiology 15 (1962) 473-493.
[3] NAVARRO L Citrus shoot tip grafting in vitro - Biotechnology in agriculture and forestry High- Tech and Micropropagation II (Y.P.S. Bajaj ed.), Springer – Verlag Berlin Heidelberg 18 (1992) 317-338.
[4] DONINI B Mutagenesis applied to improve fruit trees In: Induced mutation in vegetatively propagated plants II, proc. of Final research co-ordination meeting, Coimbatore, India, (1982) pp. 29-36.
[5] FOTOUHI GHAZIVINI R Tissue culture and frost tolerance studies in citrus spp., Ph. D. thesis, University of Salford, department of biological sciences, U K (1996) pp.
167 .