Study of Polyembryony, Seed Viability and Genetic Identification of Japansche Citroen (Citrus limonia Osbeck) seedling using SSR

Abstract

Japansche Citroen (Citrus limonia Osbeck) or JC is citrus rootstock that is often used by Indonesian citrus farmer because its wide adaptation ability and has resistant to several diseases. JC seeds should have physical, physiological, genetic and health quality. Seed has maximum quality when it is reached at the time of physiological maturity. Seed maturity, in general is decided by physical appearance of fruit, such as rind colour changes. JC seeds have polyembryony characteristic; threrefore one or more embryos per seed consist of either zygotic embryo and or nucellar embryo. Nucellar embryo can be useful for clonal propagation. Nevertheles, there were off type seedling. It is necessary to identify seedling base on morphology and molecular marker such as SSR marker. Morphology marker was easy to used and cheap, but the trully genetic must be identify with SSR. SSR could differenciate between homozygot and heterozigot genotype. The aims of this research were to acquire information about poliembryony on JC citrus, to determine physiological maturity of JC seed base on rind colour and fruit hardness, to know the effect of fruits maturity to physical and physiological quality of seed, genetic quality and number of seedling, and to identify JC seedling base on morphology marker and SSR marker. The research was conducted in Indonesian Citrus and Subtropical Fruits Research Institute from April 2012 to January 2013. The research consisted of three experiments, there were 1) study on polyembryony of JC , 2) study on effect of fruits maturity level to physical and physiological quality of seed, genetic quality and number of seedling, and 3) identification of JC seedling base on morphology marker and SSR marker. Experiment I was conducted based on visual observations on each of 30 seeds of five maturity levels. Criteria of fruit maturity are 1) dark green rind, hard fruit (> 7 kg/cm2), 2) green yellow rind, rather hard (6-7 kg/cm2), 3) pieces of yellowish green rind, rather soft (5-5.9 kg/cm2), 4) >90% yellow rind, soft (4-4.9 kg/cm2) and 5) pieces of yellow-orange rind, very soft (< 4 kg/cm2). The experiment variable were seed coat colour, the mean number of embryos per seed, polyembryony seed percentage (%) and the dominant colour of the embryo. Experiment II used randomized completely block design (RCBD) one factor. The factor is five maturity level base on fruits maturity. The criteria of fruits maturity are equal to experiment I. Each treatment was repeated three times so that there was 15 units of experiments with experimental unit consisted of 100 seeds. The experiment variable were seed moisture content (%), dry weight of 10 seeds (g), vigor index (%), seed growth rate (%/etmal), germination percentage (%), the growth percentage (%), multiple seedling percentage (%), total seedlings, off type seedling percentage (%), and total true to type seedling. Data were analyzed with analysis of variance (ANOVA). If the results obtained by analysis of variance were significant different, then tested by Duncan's Multiple Range Test (DMRT) at 5% level. Experiment III was the identification of true to type seedlings and off type seedling with morphology marker and molecular markers. This experiment used 12 samples of seedlings in experiments II and JC parent tree as a comparison. Identification of JC seedling based on descriptor list for Citrus from IPGRI with modified. Variable observed were high, the number of leaves, leaf length and width ratio, LAMI (the ratio of leaf length and width), the presence of spines, the presence of stipules and leaf shape. Identification of molecular markers with 6 SSR primers. The outline of DNA analysis with SSR markers were DNA extraction, test the quality and quantity of DNA by electrophoresis, amplification and separation of DNA by PCR, visualization of PCR results and analysis of molecular data. Bulk Segregant Analysis (BSA) was used to know the linkage between SSR markers and morphology markers. Cluster analysis of morphology marker and SSR marker used SIMQUAL procedure and SAHN method. The result showed that JC citrus has 1-6 embyo per seed. The persentage of JC seed polyembryony was 53.34 to 73.32%. The riper the seed, the more creamcoloured the dominant embryo and no green colour. JC seed physiological maturity was achieved in fruits with more than 90% rind is yellow, low hardness fruits, the color of seed skin was brownish cream and embryos color was cream. Seed maturity was not significantly different affect to the percentage of multiple seedling and percentage of off type seedling. Multiple seedling can increase the number of total of true to type seedling. Seedling leaves type, leaves shape and colour of young leaves, in general use as morphology marker of true to type and off type seedling. SSR primer AG14 which amplified in 170bp have linkage to LAMI < 0.75 but there were 40% bias. Characteristic of JC true to type seedling were single leaf type, light green shoot tip colour, elliptic leaf shape with cuncate leaf base, obtusus leaf tip and crenate leaf margins. Meanwhile, trifoliata leaf type, purplish red shoot tip colour and lanceolate lamina shape with rounded tip were an effective marker to identify JC off type seedling.