PONKAN MANDARIN ( Citrus reticulata Blanco ) IMMATURE FRUITS STORAGE Armazenamento de frutos imaturos de tangerineira Ponkan ( Citrus reticulata Blanco )

The aim of this work was to evaluate the effect of Ponkan mandarin (C. reticulata) x Pêra sweet orange (C. sinensis) immature fruits storage and sucrose concentrations on embryos in vitro culture. Fruits with 3 to 4 cm in diameter were harvested and placed inside black polyethylene bags with lateral openings and stored at 5±1oC during 135 days. Every 15 days a sample was removed, its embryos were excised and individually inoculated in test tubes containing 15 mL of MS medium (MURASHIGE & SKOOG, 1962) with sucrose (0, 1.5, 3, 6, 12, 18 and 24 g L) and 0.3 mg L GA 3 and 1 g L activated charcoal. Those treatments rested 48 hours in the dark and later in a growth room at 27 ± 1oC with a 16-h photoperiod and 32 μmol m s light intensity. Immature fruits can be stored for posterior excision and embryos culture. Fruits with 120 days after the pollination can be stored for at most 135 days without damaging the embryos viability. It was observed a better development of the aerial part and root system of plantlets from Ponkan mandarin x Pêra sweet orange embryos in MS medium with 12-18 g L sucrose.

In any breeding program, successive hybridizations are carried out in order to achieve the desired objective.In citrus breeding, immature embryos culture provides conditions for both zygotic and nucelar embryos development besides allowing an early identification of plantlets produced by zygotic embryos.The storage of fruits allow the embryos to be excised and cultivated for a long period.The low temperature during the storage delays the maturation process and the early fruit senescence.When storing seeds of Citrus paradisi Macf. in different conditions (CHACKO & SINGH, 1969), noticed that the viability was kept for more than 13 months when the seeds were stored with high humidity (above 80%), under 5-8°C.The longevity of C. karna Raf., C. jambhiri Lush., C. limonia Osb., and Rusk citrange [(C.sinensis (L.) Osb.x Poncirus trifoliata (L.) Raf.] seeds after storage was studied under environmental temperatures and 8°C, in hermetic containers, in plastic or paper bags, with or without dissecting during five months (KRISHNA & SHANKER, 1977).C. karna and Rusk citrange presented 100% seed viability, while C. jambhiri and C. limonia showed 84% and 80% viability, respectively, when stored in plastic bags containing CaCl 2 , under low temperatures.Generally the excised embryos from temperate region plants require lower temperatures than those ones from Ciênc. agrotec., Lavras, v. 30, n. 5, p. 1017-1020, set./out., 2006 plants of tropical or sub-tropical regions (NARAYANASWAMI & NORSTOG, 1964).Light must be avoided during the first days of culture, once it leads to early embryos germination.The growth of most cultures is supported by the source of carbohydrate added to the medium, which supplies metabolic energy and carbonic skeletons for the amino acids biosynthesis and production of structural proteins, and polysaccharides as the cellulose (CALDAS et al., 1998).Sucrose is the best carbon source to be used, besides offering an optimum growth, it is also a low cost source (MELLO, 1998), and for many species it is applied in the culture medium at 2-4% concentration.Below this concentration, generalized chlorosis may occur in the plantlets, and above that problems related to excessive osmotic potential of the medium may result in culture decay (GRATTAPAGLIA & MACHADO, 1990).Multiplication and growth of in vitro plants also depend on the explant and the sucrose the nitrate reductase activity, which is responsible for the cell nitrate utilization (CALDAS et al., 1998).Various concentrations are used with Citrus species for several purposes (BELOUALY, 1991;GAVISH et al., 1991).Concentrations higher than 5% have presented a good result for micrografting (NAVARRO et al., 1975).The 3% concentration presented satisfactory development to the axillary buds (PASQUAL & ANDO, 1989 a,b).The present study aimed to evaluate the effect of Ponkan mandarin (C.reticulata) x Pêra sweet orange (C.sinensis) immature fruits storage and sucrose concentrations on embryos in vitro culture.
Ponkan mandarin immature fruits obtained from crosses with Pêra sweet orange, with 3 to 4 cm diameter were used.Fruits were harvested and placed into black polyethylene bags with lateral openings and then stored at 5±1°C for a maximum of 135 days.Every 15 days a sample was removed, its embryos were excised and cultured in vitro.The embryos, independently of their development stage, were excised and inoculated in test tubes containing 15 mL of the MS medium (MURASHIGE & SKOOG, 1962) with increasing concentrations of sucrose (0, 1.5, 3, 6, 12, 18 and 24 g L -1 ), 0.3 mg L -1 GA 3 (giberelic acid ) and 1 g L -1 activated charcoal.These treatments rested 48 hours in the dark and later in a growth room at 27±1°C, with a 16-h photoperiod and 32 µmol m -2 s -1 light intensity.The storage periods were 0, 15,30,45,60,75,90,105,120 and 135 days.The plantlets variables observed were: aerial part height, number of leaves, aerial part dry matter weight and length of the roots.
The storage of fruits increased the aerial part height for all sucrose concentrations (Figure 1A).Greater number of leaves was obtained with 12 g L -1 sucrose at 90 days storage (Figure 1B).Aerial part dry matter weight was identical for 12 and 18 g L -1 sucrose at 135 days storage (Figure 1C).The dry matter weight with 18 g L -1 sucrose without storing the fruits, was significantly higher than 12 g L -1 sucrose for the same storage period.Better results for roots length were observed at 12 as well as 18 g L -1 sucrose at 135 days storage (Figure 1D).
Temperature and humidity affect the seeds longevity, either under natural environment or controlled conditions (BARTON, 1961;ROBERTS, 1972).In the fruit storing with viable embryos by more than 4 months (CHACKO & SINGH, 1969) the best conditions for seed storage of C. paradisi were high humidity and temperatures between 5 and 8°C.According to our results, this high embryo viability value after 135 days of storage can also be attributed to the absence of light, once the fruits were being stored.Under these conditions the early embryo germination was avoided which turn possible them to complete their development.The temperature utilized for the fruits storage was also an important factor in preserving the embryo characteristics.The fact that in a same fruit one can find embryos at different stages of development, and that they consequently have different requirements concerning the osmotic potential (SHARMA et al., 1996), a greater sucrose demand would be needed in order to avoid the embryos early germination.In this paper intermediate sucrose concentrations (12 to 18 g L -1 ) showed the best results.
Fruits with 120 days after the pollination can be stored for at most 135 days without damaging the embryos viability.

FIGURE 1
FIGURE 1 Aerial part height (cm) -A, Number of leaves -B, Aerial part dry matter weight (mg) -C, Root length (cm) -D, of plantlets from Ponkan mandarin [(Citrus reticulata Blanco) x Pêra sweet orange (C.sinensis (L.) Osb.] in vitro culture immature embryos, with different sucrose concentrations from stored fruits.