In Vitro Conservation Of Apple And Other Fruit Crop Germplasm

Plant genetic resource is one of the most essential substance bases for human to survive and develop, and to keep its diversity is the important guarantee for sustainable development. Fruit trees are important economic crops, and play an important role in national economy. Planting in open field is the traditional conservation method for fruit tree germplasm. But disease and insect pests, natural disasters and shortage of fund often trouble this method, which even leads to the loss or facing loss of some germplasm. In vitro conservation has been becoming the important complementary conservation technique with its characteristics of safety and saving room and money. Based on pre-researches, further studies were carried out on important fruit trees such as apple, pear and pomegranate. The results are as follows:1 In vitro cultured shoots of fifteen pear cultivars from four pear species were established including Pyrus communis, P. pyrifolia, P. bretschneideri and P.sinkiangensis, and MT+BA1.0 mg/L+NAA0.1 mg/L+GA32.0 mg/L was determined as the common subculture medium suitable for the above species, as provided materials and basic technique for in vitro conservation of pear germplasm. Based on the principles of plant virus elimination by micro-shoot tip culture and heating treatment, internal bacteria were successfully eradicated by the following protocol. In vitro pear shoots were cultured in dark to accelerate the shoots elongation and weaken the tubular conductive structure, and then the yellowed shoot tips were subcultured. This is a new method to get internal bacteria free materials in vitro from matured explants growing in the field.2 Single somatic embryoid lines of six apple genotypes including Gala, Orin, Fuji, Balenghaitang, zumi and MM₁06 were established by embryoid regeneration from leaves, which provided materials for the somatic variation studies.3 Cochicine treatment for a long time added in the medium was determined as the better method to double chromosome of pomegranate, and tetraploid pomegranate plants were first produced by this method. Tetraploid plants exhibit the characteristics of darkgreen leaves, bigger flowers and reduced pollen viability. As provided a new method for pomegranate breeding.4 Approaches for in vitro conservation of three kinds of fruit crops were established by slow growth method at normal temperature by the adjustment of in vitro shoots status, the selection of explants and its size, reducing oxygen supply and water evaporation through seal. In vitro apple shoots could be conserved for 12 to 25 months, pear and pomegranate shoots for more than 7 months without subculture by this method.5 Approaches for in vitro shoots conservation of different apple and pear genotypes at low temperature were established and optimized. And the survival rate of apple and pear shoots being conserved at 3 + 0. 5 °C for 28 months and 24 months respectively could be close to 100%. The approaches for cryopreservation of apple shoots were optimized by pretreatment with glycerol and ABA.6 The effect of explants and nitrogen level in the medium on in vitro flower formation of pear current shoots was studied. Bourse shoots displayed a higher frequency of flower formation than primary vegetative ones; and increasing the carbon and nitrogen ratio through lowing the nitrogen level in the medium could enhance flower differentiation. The flower differentiation rate of bourse shoots cultured on 1/3MS-N could reach 60% compared to 15% of primary vegetative shoots.7 Flower differentiations was observed when pear shoots were subcultured after conservation on the medium with high carbon and nitrogen ratio at low temperature. And flower differentiation rate of shoots cultured on the conservation medium with 1/2MS macroelement and lOOg/L sucrose reached 80%. The high frequency flower formation system could provide the technique platform for flower differentiation and molecular development of flower organ studies.8 Single embryoids from apple leaves were tested by flow cytometer and AFLP to examine the genetic stability. No ploidy variation was found and all the 104 single embryoid lines remained diploid consistent with the parent. But AFLP polymorphism was found among the tested 29 single embyoid lines out of 104. The variation rate based on bands was 0.099%. The same varied bands were found among different single embyoids, which show that hot mutation spot existed.9 Flow cytometer and AFLP markers were adopted to detect the ploidy and DNA variation of a single-embryoid derived line of Fuji apple conserved by slow growthmethod and cyopreservation respectively. No ploidy variation was found in the shoots conserved by the above two method. But AFLP fingerprints varied at a frequency of 0.190% and 0.387% based on bands respectively.10 Two of the varied bands were reamplified, sequenced and functional analyzed. Both the two bands covered codon domain sequence. The 250bp fragment, significant homology to partial sequence of Arobidopsis, Oryza and May genomics, covered several frames, and the homologous sequence from chromosome 5 of Oryza belongs to partial codon domain sequence of sorting nexin-like protein gene family. The results displayed that the two variations could influence the protein translation, and the 250bp fragment could be partial codon domain sequence of a gene family.