Application of Biotechnology on Apple Breeding

Xueying LI Jie HAO Xinmin YAN Xian’ge WANG Jianzhong FENG Xiangmin SUO

Abstract Modern biotechnology is a kind of potential technology. The advantages of this method have aroused much attention. Recently， biotechnology has evolved very fast in apple tree， and has got much progress in production and breeding of seedlings. This paper summarized the advances in tissue culture， molecular marker and gene engineering. The emphases and direction which need further investigation were also discussed.

Key words Apple; Biotechnology; Tissue culture; Molecular marker; Gene engineering

Biotechnology refers to an interdisciplinary technology which uses the biological system and applies advanced biological and engineering techniques to process substrate materials or not， to provide various products required or for a specific purpose. It is a product of the high development and mutual interpenetration and combination of modern biology， microbiology， biochemistry， genetics， cell biology， especially molecular biology and molecular genetics， which is also closely combined with modern experimental techniques， engineering techniques and electronic computers. Biotechnology has been widely used in many fields such as rapid propagation， germplasm preservation， variety identification， new variety breeding and transgenic of apples[1-2].

Application of Tissue Culture in Apple Breeding

Apple tissue culture has been able to regenerate plants from different levels such as plants， organs， tissues and protoplasts. Tissue culture has shown great potential in rapid propagation， purification of hybrids， distant hybridization breeding and shortening of breeding cycles[3-6].

Invitro asexual reproduction technique

Invitro asexual reproduction is a method which applies sterile culture technique to culture organs such as shoot tips， axillary buds， leaves， scales， roots and bulbs from excellent plants and their tissue sections in vitro[7-10]， to obtain a large number of individuals with accordant hereditary in a short term. In 1973， Jones et al. successfully cultured the stem tips of apple rootstocks M26 and M9. Later， Button et al. from the United States used this technique to produce the apple rootstock M27 bred by the East Malling Research Station in the United Kingdom， which promoted its promotion and application. China has also done a lot of work in this area， which accelerates the promotion and application of new apple varieties. In addition， due to the strict treatment of invitro technology， it can remove insects， common fungal diseases and some bacterial pathogens and viruses easily[11]， and is thus an effective measure for rejuvenating varieties. The viruses and diseases that have been successfully removed include apple chlorotic leaf spot virus， mosaic virus and ring rot disease.

Embryo culture technique

Embryo culture can allow the development of embryos that are underdeveloped in breeding for early maturity or embryos that have stopped development in distant hybridization to continue， thereby increasing the chance of breeding very early maturing varieties or overcoming obstacles such as distant hybrid infertility and hybrid dysgenesis to obtain distant hybrids. Endosperm culture opens up a new way for cultivating triploid plants and selecting ploidy variants[12]. Sun et al.[13]performed hybridization using Fuji apple as the female parent and "Golden 20th Century" pear as the male parent， and the fruit setting rate reached 4.183%; and embryo culture was carried out 60 d after the hybridization， and sprouting was successfully induced.

Protoplast culture technique

Protoplasts are "wallless cells"， which， through directly ingestion of foreign DNA or organelles or heterogeneous protoplast fusion， can create somatic hybrids which can be directly used for genetic improvement of plants， and have great potential for production and application. Apple is a tree species which is studied earlier for protoplast isolation and culture in fruit trees. In 1983， Niizeki et al.[14]isolated protoplasts from the anther calli of the apple cultivar Orel for the first time， and obtained calli after culture. In 1988， PatatOchatt et al.[15]reported for the first time that regenerated plants were obtained by culturing mesophyll protoplasts of three apple genotypes. Since then， there have been some reports about plants regenerated from apple protoplasts[16-20]， and more than a dozen apple genotypes of protoplasts have been cultured to obtain regenerated plants[21]. So far， in the study of apple protoplast technology， there has been no major obstacle from the separation of protoplasts to the formation of calli， and the most critical problem is that the differentiation of calli to regenerated plants is more difficult， which is the bottleneck restricting the wide application of apple protoplasts. Apple protoplast research is now situated at the stage of tackling key problems[22].

Protoplast fusion includes somatic hybridization and cytoplasmic hybridization. Protoplast fusion can overcome hybridization incompatibility between distant species and different genera. It can also overcome the problem that the sexual reproduction process cannot be completed due to abnormalities of reproductive organs in distant species and different genera; it can also improve the traits controlled by plasmagenes; and the protoplast fusion of the diploid protoplasts and the protoplasts of pollen haploids can give the triploids of seedless fruits， which is a new way to breed seedless varieties of fruit trees. James et al.[23-24]first studied the fusion of apple protoplasts and saw the formation of heterokaryons， but no heterokaryon division was observed. Saito et al.[25]applied PEGDMSO method to the study of apple fusion， which can form heterokaryon at low frequency， and finally give calli after culture， but the calli were not identified as a fusion bodies. He et al.[26]fused haploid protoplasts of Gala and Fuji apples under the induction with PEG， and obtained after about 60 d of culture， calli， which were induced to differentiate and regenerate plant RH21， which was identified to be fusion plant having the characteristics of both parents.

Application of molecular marker technology in apple breeding

Apple has the characteristics of long juvenile phase， long growth cycle， large plant， high degree of heterozygosity of the genome and selfincompatibility， and its traditional genetic breeding consumed a lot of time and labor with high cost. With the rise of molecular biology in recent years， especially the development and application of DNA molecular marker technology in apples， molecular marker technology has become an effective research method and auxiliary breeding method in apple genetic breeding research. The molecular marker is kind of genetic marker that directly reflects genetic variation at the DNA level and has the characteristics of enough environmental and material sources， no effect on phenotype and rapid detection and easy operation. Therefore， in the past few years， there have been rapid developments. So far， dozens of molecular marking techniques have emerged， mainly in three categories：  RFLP （Restriction Fragment Length Polymorphism） as a representative developed on the basis of conventional southern hybridization，  RAPD （Randomly Amplified Polymorp hicDNA）， STS （Sequence Tagged Site）， SCAR （Sequence Characterized Amplified Region）， AFLP （Amplified Fragment Length Polymorphism） as representatives developed on the basis of PCR， and  SSR （Simple Sequence Repeat） as a representative developed on the basis of repetitive sequence. The molecular markers often used in apple genetic breeding research are also mainly RFLP， RAPD， AFLP， SSR and SCAR[27].

Variety identification

Apples have the characteristics of wide geographical distribution， high gene heterozygosity and large variation population， and it is difficult to accurately study and analyze them in the past. DNA molecular markers detect differences at the genomic DNA level， and can be used to compare varieties with the help of genetic maps which can cover the entire genome， thereby greatly improving the reliability of the results. DNA molecular markers can be used for identification， preservation and management of germplasm resources.

DNA molecular marker technology can essentially reflect the differences between biological individuals， and can carry out parentoffspring pedigree analysis， and determining the genetic relationship and evolutionary status of apples is beneficial to exsitu collection of germplasm resources and the insitu collection and preservation of wild apple resources. And DNA molecular marker technology can identify the core germplasm and represent the genetic diversity of resources to the largest extent with minimal amounts of germplasm resources. In 1993， Koller et al.[28]successfully distinguished 11 apple varieties with primer P （5′ACGAGGGACT3′） applying RAPD labeling technique. Gianfranceschi et al. used SSR marker technique to distinguish 19 apple varieties with only two SSRs. Harad et al.[29]determined by RFLP and RAPD techniques that the male parent of Tsugaru apple is Jonathan apple， the parents of triploids Jonagold （Golden Delicious≠Tsugaru） and Mutsu （Golden Delicious≠India） are diploids， and further analysis suggested that the female parent Golden Delicious provides diploid gametes for its offspring during meiosis. Hokanson et al.[30]detected 66 apple lines using eight SSR primers to determine genetic identity and estimate genetic stability. The results showed that variations were found in the 66 lines at a high level， and the genetic identity data produced a genetic correlation phenogram， which was confirmed by geographical origin and information about the family.

Gene mapping and cloning

Gene mapping and cloning play an important role in the study of important agronomic traits in apples. Gene cloning has important applications in genetic markers and genome mapping. The understanding of the basic functions of many important genes can only be deepened after cloning， and these genes can be modified then to create new phenotypes and achieve their transfer between different varieties or species. During gene cloning， genetic markers linked to the target gene are screened， followed by highresolution mapping using the screened markers to determine molecular markers closely linked to the target gene， and largelength genomic libraries （YAC library， BAC library， etc.） are screened using markers closest to the trait as a probe; and then， clones associated with the marker gene are identified， and through subcloning and genome walking， large fragments containing the gene of interest are obtained and finally verified by transformation and a complementation test. Molecular markers linked to important traits have been found in apples， such as the antiscab disease gene Vf[31-32]， the antidowny mildew gene Pl1[33]， the pericarp color gene Rf[34]， the top bud fruiting gene Tb， the flower bud differentiation gene Rbb， the root sucker occurrence gene Rs[35-37]and the columnar trait gene Co[38-39]. Some of these markers have been successfully transformed into RAPD and SCAR markers， and with the deepening and development of molecular marker research， more genes controlling apple agronomic traits will be labeled and cloned[40].

Markerassisted selection

In apple breeding， early selection is important. Molecular markerassisted selection is the indirect selection of target genes through genetic markers， during which some undesired traits can be discovered and eliminated in time. It can greatly improve the accuracy of selection and improve breeding efficiency. Molecular markerassisted selection is mainly used in apple breeding for the selection of hybrid parents， early selection of hybrid seedlings， tracking of chromosome segments， genetic testing and direct screening of multiple disease resistance traits. The selection of parents is a key link in cross breeding， while the selection of parents according to the phenotypic characteristics of breeding materials is often interfered by environmental conditions with low selection efficiency. Molecular markers can assist the selection of the optimal parent combination with favorable genes when avoiding adverse genes， and the population size and selection intensity of hybrid progenies can be calculated. Consequently， the selection of parents is more rapid and accurate， and the breeding efficiency is improved thereby. There are two main methods for molecular markerassisted selection. One is to determine the presence or absence of the target gene by detecting the presence or absence of molecular markers closely linked to the gene of interest. The second is to establish a highdensity molecular genetic map and to perform selection with the two molecular markers on both sides of the target gene. Molecular markerassisted selection has been widely used in the study of some important agronomic traits， such as scab disease resistance， aphid resistance， powdery mildew resistance， dwarf denseplanting trait， fruit color， salt tolerance and fruit acidity. Cheng et al.[41]have used the RAPD markers linked to the Thd2I gene to predict the fruit color of apple seedlings. In apple breeding for scab resistance， RAPD markers closely linked to the Vf gene have been widely used. Molecular markerassisted breeding makes early selection， prediction and directed change of apple traits possible， which greatly speeds up the process of apple breeding， improves the efficiency of breeding and overcomes the problem of long period restricting apple breeding in the past.

Genetic Engineering and Apple Breeding

Genetic engineering， also known as recombinant DNA technology， refers to technique which inserts a gene of interest obtained by DNA cloning technology into a virus， plasmid or other carrier molecules， constructing a new combination of genetic materials， which is introduced into a host cell or individual that does not have such a molecule， but expresses it continuously and stably then， producing a new individual needed by human. This combination of this technology with breeding produces molecular breeding. Genetic engineering opens up new way for apple breeding which has important theoretical and practical significance[42-43]. Once transgenic apple varieties are acquired and the target genes are expressed as expected， they can be propagated in large numbers through asexual reproduction such as tissue culture， grafting or cuttings[44].

In apple breeding research， genetic engineering is most widely applied to improvement of disease resistance and insect resistance[45-46]， during which the exogenous genes or DNA is introduced into apple cells through the Agrobacterium tumefaciens 2Ti plasmid method， PEG （polyethylene glycol） method or particle bombardment method. The transformation of apple insectresistant genes succeeded first in the transformation of Bt gene into Greensleeves variety. James et al. （1987） transformed A. tumefaciens with vector "Binb" into apple leaves， and the regenerated plants expressed the nopalineproducing and antikanamycin genes. Mariotti et al. （1987） transformed genes encoding the auxin and agroprote in root plasmids into the apple rootstock with difficulties in rooting， making rooting of the rootstock easy. Shi et al.[45]transformed the cowpea trypsin inhibitor gene （CpTI） initiated by the highefficiency promoter to Fuji， Jonagold， Wang Lin and Gala apples using the A. tumefaciensmediated leaf disc transformation method， and it was proved that all the four varieties were transformed and regenerated.

Although the apple transformation system has been established and the marker genes have been transformed into the apple and expressed， it is not easy to isolate valuable genes or transform genes， and it is difficult to culture in vitro， as the fruit tree is genetically complex and the important traits are controlled by multiple genes. Gene transformation is still some distance away from practical applications， but we can see the dawn from many successful examples. With the continuous advancement of science and technology， the application of genetic engineering to directly manipulate apple traits gradually matures， which will definitely play a huge role in the improvement of fruit cultivars.

Conclusions

The booming modern biotechnology has been recognized as a technology with great potential. Combined with conventional breeding techniques， the process of apple breeding has been greatly accelerated， and the breeding efficiency is improved. As a result， the problem of long apple breeding cycle in the past is overcome. In view of the current development of Chinaюs apple industry， the development of biotechnology in the near future should focus on economically applicable technologies， which could create higher economic benefits. For instance， combining embryo culture with conventional breeding overcomes problems in distant hybridization and creates new germplasm resources. The development of economical and practical molecular marker DNA fingerprinting technology and early identification technology of hybrids further accelerates the breeding pace and ensures the quality of breeding. Molecular markers are combined with transgenic technology to screen and clone valuable genes which are used for improving apple traits by transgenic technology.

References

[1]MA Y， CHAI CJ. Application of biotechnology in fruit trees[J]. Northern Fruits， 2004， （1）： 1-3. （in Chinese）

[2]WANG ZY， WANG Y， DONG DZ. Application of biotechnology in fruit trees[J]. World Agriculture， 1997， （12）： 20-23. （in Chinese）

[3]PAN ZG， DENG XX. Research progress on apple tissue culture regeneration technology[J]. Fruits Science， 1998， 15（3）： 261-266. （in Chinese）

[4]PAN ZG， DENG XX， ZHANG WC. The development of protoplast research in apple[J]. Acta Horticulturae Sinica， 1997， 24（3）： 239-243. （in Chinese）

[5]SHI XX， GAO Y， MA BK， et al. Establishment of sterile propagation system in apple shoot tip culture[J]. Journal of Agricultural University of Hebei， 1997， （02）： 45-49. （in Chinese）

[6]DU XM， LI DK. Application of growth regulators to apple tissue culture[J]. Chinese Agricultural Science Bulletin， 2002， 18（4）： 75-79. （in Chinese）

[7]HAN Y， WANG GL， XING Z， et al. Tissue culture and rapid propagation of ornamental apple "Hongxia"[J]. Plant Physiology Communications， 2005， 41（2）： 188. （in Chinese）

[8]XU SY， BAI HX， GUO HL， et al. Study on tissue culture and rapid propagation technology of highacid apple[J]. Shanxi Fruits， 2006， （1）： 6-8. （in Chinese）

[9]ZHENG YJ， YAO HY. Studies on tissue culture technology of GM256， a dwarf rootstock of apple[J]. Journal of Jilin Agricultural Sciences， 2008， 33（1）： 26-27. （in Chinese）

[10]ZHOU HC， ZHAO X， ZHAN YW. Tissue culture and rapid propagation of Malus Teli[J]. Inner Mongolia Agricultural Science and Technology， 2008， （2）： 72-73. （in Chinese）

[11]WANG JX. Research and application of apple virusfree technology[J]. Northern Fruits， 1994， （2）： 2-4. （in Chinese）

[12]WU XJ， LIU SQ. Vegetative reproduction and chromosome ploidy of apple endosperm plants[J]. Acta Horticulturae Sinica， 1981， 8（3）： 11-14. （in Chinese）

[13]SUN L， LENG P. Applepear hybrid and invitro rescue culture of zygotic embryo[J]. Journal of China Agricultural University， 2008， 13（2）： 25-29. （in Chinese）

[14]NIIZEKI M， HIDANO Y， SAITO K. Callus formation from isolation protoplasts of apple， Malus pumila Mill[J]. Japan J Breed， 1983， 33： 369-374.

[15]PATATOCHATT EM， OCHATT SJ， POWER JB. Plant regeneration from protoplasts of apple rootstocks and scion varieties（Malus≠domestica Borth.）[J]. Plant Physiol， 1988， 133： 460-465.

[16]DING AP， WANG HF， CAO YF. Plant regeneration from cotyledon and cell suspension protoplasts of apple （Malus domestica cv. Starkrimson）[J]. Plant Cell， Tissue and Organ Cultrue， 1995， 400： 145-149.

[17]HUANCARUARUNAPERALES E， SHIBATE M. Protoplasts culture and regeneration of plant of Malus genotypes by means of alginate embeding[J]. Plant， 1991， 82： 15.

[18]HUANCARUNAPERALES E， SHIBATE M. Plant regeneration from leaf protoplasts of apple[J]. Plant Cell， 1993， 34： 71-76.

[19]PATATOCHATT EM， BOCCONGIBOD J， DURON M， et al. Organogensis of stem and leat protoplasts of a haplold golden delicious apple clone （Malus≠domestica Borkh.）[J].Plant Cell Reports， 1993， 12： 118-120.

[20]HE DY， SUN S. plant regeneration from protoplasts of haploid induced by irradiation in apple[J]. Acta Agriculturae Nucleatae Sinica， 2004， 18（6）： 411- 415. （in Chinese）

[21]WANG XJ. Study on protoplast isolation technology of apple rootstock[D]. Baoding： Agricultural University of Hebei， 2000. （in Chinese）

[22]XIAO JH， SUN JS. The development of protoplast research in apple[J]. Journal of Agricultural University of Hebei， 2002， 25（Sup.）： 98-100. （in Chinese）

[23]JAMES DJ， PASSEY AJ， MALTOTR SB. Isolation and fusion of protoplasts[R]. Rep E Malling Res Station for 1983， 1983：63-65.

[24]JAMES DJ， PASSEY AJ. 1Protoplast culture and the use of apomixis in vitro for the regeneration of fruit plant. In： Plant Tissue Culture and its Agriculture Application[C]. Butterworths， Sevenoaks， Kent， 1986， 349-357.

[25]SAITO A， NIIZEKL M， SAITO KI. Organ formation from cali and protoplast isolation culture and fusion in apple， Malus pumila Mill[J]. Japan Soc Hort Sci， 1989， 58： 483-490.

[26]HE DY， CHEN ZG. Somatic hybrids through protoplasts fusion in apple[J]. Journal of Fujian Agriculture and Forestry University， 2000， 29 （1）： 128. （in Chinese）

[27]LI YH， HAN ZH， XU XF. Application of molecular marker technique in apple breeding[J]. Biotechnology Bulletin， 2002， （6）： 11-13. （in Chinese）

[28]KOLLER BL， EHMAN A， NCDERMOTT JM， et al. Identification of apple cultivars using RAPD markers[J].Theor Appl Genet， 1993， 85： 901-904.

[29]HARADA T， SATA T， LSHIKAWA R， et al. DNARAPDs detect genetic variation and patermity in Malus[J].Euphytica， 1993， 65（2）： 87-91.

[30]HOKANSON SC， SZEWCMCFADDEN AK， LAMBOY WF， et al. Microsatellite （SSR） markers reveal genetic identities， genetic diversity and relationships in a Malus domesticaborkh core subset collection[J]. Theor Appl Genet， 1998， 97： 671-683.

[31]KING GL， ALSTON FH， BROWN LM， et al. Mutiple field and glasshouse of scab resistance in apple[J].Theor Appl Genet， 1998， 96： 699-708.

[32]YANG HY， KORBAN SS， KRUGER J， et al. A randomly amplified polymorphic DNA （RAPD） marker tightly linked to the scabresistance gene Vf in apple[J].Amer Soc Hort Sci， 1997， 122（1）： 47-52.

[33]MARKUSSEN T， KRUGER J， SCHMIDT H， et al. Gene Pl1 from Malus robusta in cultivated apple[J]. Plant Breeding， 1995， 114： 530-534.

[34]LANDRY BS， CHENG WJ. Phylogeng analysis of 25 apple using RAPD markers and tactical gene tagging[J].Theoretical and Applied Genetics， 1999， 89： 847-852.

[35]WEEDEN NF， HEMMAT M， LAWSON DM， et al. Development and application of molecular marker linkage map in wood fruit crops[J]. Euphytica， 1994， 77： 71-75.

[36]WEEDEN NF. Genetic studies in pea and apple――the yin and yang of mapping in plants[J]. Hortscience， 1996， 31： 1111-1116.

[37]WEEDEN NF， HEMMAT M， LAWSON DM， et al. Development and application of molecular marker linkage maps in woody fruit crops[J].Euphyicca， 1994， 77： 71-75.

[38]PADGETTE SR， KH KOLAC Z， X DELANNAY， et al. Development， identification and characterization of a glyphosatetolerant soybean line[J].Crop Sci， 1995， 35： 1451-1461.

[39]BING DJ， DOWNEY RK， RAKOW GFW. Assessment of transgene escape from Brassica rapa （B. campestris） into B. nigra or Sinapis arvensis[J]. Plant Breeding， 1996， 115： 1-4.

[40]YAO YX， ZHAI H. A review of molecular studies on apple genome[J]. Journal of Fruit Science， 2004， 21（6）： 586-591. （in Chinese）

[41]CHENG FS. Development of a DNA marker for Vma gene conferring resistance to apple scab[J].Theoretical and Applied Genetics， 1996， 89： 847-852.

[42]CHEN JS， DENDEKAR AM， URATSU SL， et al. Preliminary report on apple transgenic technology[J]. Acta Horticulturae Sinica， 1992， 19（2）： 101-104. （in Chinese）

[43]ZHAO H， SONG GY， ZHANG GZ， et al. Study on introducing kidney bean chitinase into apple rootstock[J]. Acta Laser Biology Sinica， 1998， 7（3）： 163-167. （in Chinese）

[44]SHI BH， FU YM， ZHAO ZY， et al. Study on invitro culture of apple leaves[J]. Acta Botanica BorealiOccidentalia Sinica， 1995， 15（1）： 67-73. （in Chinese）

[45]SHI XX， WANG B， DU GQ， et al. Studies on Agrobacteriummediated CpTI gene transfer in commercial apple cultivars[J]. Acta Horticulturae Sinica， 2000， 27（4）： 282-284. （in Chinese）

[46]LIU QZ， ZHAO HJ， LIU P， et al. Regeneration of tetraploid plants with cecripin MB39 gene from‘Royal Gla’apple[J]. Acta Horticulturae Sinica， 2001， 28（5）： 392-398. （in Chinese）