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Chapter – II
Review of Literature
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Tissue culture work on apple was initiated by Letham (1958). His
pioneering experiments on apple fruit tissues enabled him to get callus on
White’s medium (1943) supplemented with 2,4-D (6.0 mg/l). Nitsch (1959)
and Saad (1964, 65) made similar attempts and obtained callus but failed to
differentiate it. Nitsch (1959) used Gouthret’s nutrient medium but Saad
(1965) and Saad and Brome (1964) used different salt solutions and recorded
nutritional and temperature requirements of apple callus grown in vitro. Jones
(1967) started extensive work on shoot tips of M.26 and M.7 root stocks of
apple and suggested that 60,000 green shoots can be produced from single
shoot apex in 8 months. Zimmerman (1984) developed explants and discussed
various advantages of the technique over traditional cultivation practices.
Achievements made on in vitro apple culture from 1958 to 2010 are quite
encouraging (Table 3). The successful results achieved by various workers in
different apple cultivars and root stocks by using different explants in vitro are
discussed in detail below.
I. Shoot Apex Culture
Tissue culture work on shoot apices of apple was started by Jones
(1967). He cultured shoot apices M.26 and M.7 root stocks in vitro on Knop’s
solution (1922/1865) with minor salts of MS (1962) and BA (1 mg/l) and
observed that buds grow much better in rotating culture medium if they were
first placed for about 10 days to two weeks under stationary conditions.
However, he reported no effect on shoot proliferation. Pieniazek (1968)
extended the results of Jones to shoot apices of 5-year old Antonovka apple
seedlings and observed that BA stimulated growth of lateral and collateral buds
on shoot tips. Cernenko and Strelikov (1968) reported proliferation of apical
buds of apple on simple salt solutions gelled with agar. Messer and Lavee
(1969) observed that the growth characteristics of callus correspond to the
relative vigour of mother plants. Powel (1970) also reported BA to be
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TABLE 3: Achievements made on in vitro apple culture from 1958 - 2010.
Cultivar Explant Culture media Phytohormones mg/l Results References
Cox’s Orange Pippin, Sturner Pippin, Granny Smith
Fruit tissue
White’s medium + Myo inositol (0.2), Biotin (0.2), Ca panthothenate (0.2)
2,4-D (6.0)
Callus Letham, 1958
Golden Delicious Belle de Boskoop
Fruit tissue
Gautheret medium + Myo inositol (100), Ca panthothenate (40),
Thiamine (10)
----- Callus Nitsch, 1959
Cortland McIntosh Apple Callus W – 63 (White’s) IAA, BA Callus Saad and Broome 1964
Cortland McIntosh Stem, leaf, petiole and fruit
W – 63 (White’s) IAA, BA Callus Saad, 1965
M 26 & M.7 Meristem, Shoot apices
Knop (Macro) + MS (Micro), Na Fe EDTA (20)
BA (1.0)
Axillary shoots and roots
Jones, 1967
Antonovka Axillary buds MS BA Proliferation and growth Pieniazek and Jankiewiez, 1966
Malus pumila Stem and root tissues MS –– Callus Cernenko and Strelikovo, 1968
Antonovka seedlings Shoot tips MS + Myo inositol (100), BA Normal growth Pieniazek, 1968
M 13, M.9 Shoot tips MiS (Millar & Skoog) x2 IAA (2.0), Kn (0.2) Callus Messer and Lavee, 1969
Name not given Fruit Several media ––
Callus Nitsch et al ., 1970
Cortland Northern Spy seedlings
Buds DP (Dutcher & Powell) BA (0.1-1.0) Callus, axillary buds, normal growth
Powell, 1970
M 16 and 3-430 Callus W-63 (White’s) 2X NAA (2.0), Kn (0.2) Callus Lavee and Hoffman, 1971
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Ralls, Jonathan and Fuji Anthers Millar’s medium 2,4-D, IAA and Kn Roots and buds Nakayama et al., 1971,72
Cortland seedlings, McIntosh, Red delicious
Shoot tips MS NAA (2.0), Kn (1.25)
Callus Chong and Taper , 1972, 1974a and 1974b
Cortland Northern Spy seedlings
Axillary buds DP (Dutcher & Powell) BA (0.1-1.0) Multiple shooting Dutcher and Powell, 1972
Granny Smith Shoot tips EL (Elliott) BA (0.23) or Zeatin (0.22) Leafy shoot Elliott, 1972
Golden Delicious Koko and
Malus prunifolia Borkh
Stem
FN (Fujii and Nito) + Myo inositol (0.1), Biotin (0.01),
Ca penthothenate (10), Pyridixine (1.0)
-----
Callus Fujii and Nito, 1972
McIntosh seedling Axillary buds
MS + Myo inositol (100), Glycine (5.0), Thiamine (0.4), Nicotineamide (1.0) Pyridixine (1.0)
IAA (57.0),
Kn (1.25)
Shoots and roots Walkey, 1972
Cox’s Orange Pippin,
M 2 root stock Shoot apices
Knop (Macro) + MS (Micro),
Na Fe EDTA
BA (1.0)
Shoot proliferation Jones, 1973
MM.111, MM.106, M 26, Jonagold, Mutsu,
Meristem MS+Glycine (5.0), Myo-inositol (100.0), Thiamine (0.4)
IAA (10.0), BA(1.0), GA3(0.1)
Axillary bud and root Quoirin, 1972, 74 & 78
Cox Orange Pippin Shoot tips LS + Thiamine (1.0), Myo inositol (100.0)
2,4-D (0.5), Kn (0.1-1.0) Callus Abbott and Whitely, 1974 & 76
Malus pumila Shoot apices MS PVP Control of oxidative browning
Walkey, 1974
Malus pumila Callus MS IAA Rooting Epistain et al ., 1975
Jonathan Anther MS IAA (10.0), Kn (1.0) Callus, Embryoids Kubicki et al ., 1975
Golden Delicious Fruit MS + Myo inositol (100), Biotin (0.01), Ca panthothenate, Thiamine
2,4-D (1.0), BA (0.1) Callus Pech et al., 1975
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Niedzwetzkyana Shoot tips MS + Myo inositol (100.0), Glucine (2.0)
NAA (2.0), Kn (0.1-1.0) Callus Coffin et al ., 1976
M7, M26 root stock Shoot tips MS+ Thiamine (0.4), Myo inositol (100.0)
Phloroglucinol (PG),
Phloridzin (PZ), IAA (10), BA (0.5-1.0), IBA (1.0), GA3 (0.1)
Shoot proliferation, rooting
Jones, 1976
M.7 root stock Shoot tips K (Knudson) +MS(micro)
Phloroglucinol (PG)(160), Pyrogallol, Caffeic acid, Catechol, IBA(10.0)
Multiple shooting, rooting Jones and Hatfield, 1976
M.26 root stock Shoot tips MS BA Axillary shoot proliferation
Hopgood and Ferrel, 1977
M.26 Shoot tips MS+ Na Fe EDTA(20), Myo inositol (100.0)
PG(162), IBA (1.0), GA3 (1.0)
Rapid multiple shoots, rooting
Jones et al ., 1977
Jonathan Anther MS IAA (1.0), Kn (1.0) Callus, Embryoids Milewska-Pawliezuk and Kubicki, 1977
Jonathan and McIntosh Endosperm MS –– Callus, Complete plantlet Mu Shih-Kin et al., 1977
M.26 Shoot tips WPM IAA, BA, GA3 Axillary bud and root Quorin et al., 1977
Lord Lambourne Lord Derby
Stem cuttings MS IBA (50) Multiple shooting Robinson and Schwabe, 1977 (a &b)
Lodi, Golden Delicious, York Imperial
––
MS + Myo inositol (100), Biotin (0.01),
Ca panthothenate (1.0), Thiamine (1.0), Nicotineamide (1.0), Pyridixine (1.0)
2,4-D (1.0), BA (0.1)
Callus Wallner, 1977
Malus pumila Shoot tips ----- ----- Microgrfting Alskief and Villemur, 1978
Root stock Antonovka KA
313, M.9, EMLA-9 Shoot tips MS ----- Shoot proliferation Cheng, 1978
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Malus pumila Shoot tips, stem cuttings
----- ----- Rooting Child and Higes, 1978
Jonathan Meristem KN (Kundson)
FeSO4.7H2O (27.8), Glycine (2.0)
NAA (1.1), BA (8.9) Axillary bud proliferation Huth, 1978
McIntosh seedlings Meristem MS + Myo inositol (100), Glycine (2.0)
BA, NOA, GA3
Axillary bud proliferation Lane, 1978 & 82
Golden Delicious Seedlings Leaf, cotyledons, Hypocotyl
MS with B5 vitamins BA (10.0), NAA (3.0) Embryoids and intact
plants Liu et al., 1978
Malus pumila
Embryo, Stem segments
F + Myo inositol (100), Glycine (2.0)
----- Callus Schneider et al., 1978
Malus asiatica and Malus prunifolia
Shoot tips MS ----- Differentiation of plantlets Wang and Sui, 1978
Jonathan cultivar Shoot tips MS(1/2) medium with perlite-vermiculite (1:1)
IBA (0.5-1.5µM) or NAA (0.5-1.6µM)
Plantlet formation Zimmerman, 1978
Variety not mentioned Fruit tissues MS + Sorbitol, CaCl2,
Chloramphenicol –– Callus, protoplasts Anderson et al ., 1979
M.9 root stock Stem segments MS BA (2.2), NAA (10.7) CH, IBA Plantlet formation Chen et al ., 1979
Golden Delicious Shoot tips MS + Myo inositol (100), Glycine (2.0)
NAA (10), BA (10) Callus and embryiods Eichholtz et al ., 1979
Granny Smith Fruit (Mesocarp) MS+ Thiamine(0.1), Pyridoxine (0.1),
Niacin (0.1)
2,4-D (2.5), BA (5.0) Callus Faye and Wayne, 1979
M.9 Shoot tips LS BA (2.0), IBA (0.1),PG Rooting James and Thurbon, 1979
Malling King, M.25,M.26 Malling Suntan, James Grieve, and Cox’s Orange Pippin
Shoot tips MS MS+BA Lateral shoots, complete
plantlets Jones et al. , 1979
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Golden Delicious Shoot tips MS + thiamine(0.1), Pryridoxine(0.5)
BA (5.0) Normal growth Lundergan and Janick, 1979
Golden Delicious
Seeds, cotyledons,
leaves, embryos, roots, shoot tips, and fruit tissue
MS, White’s (BW) +sucrose (2%) Casein Hhdrolysate (CH) (100)
NAA (4.0), Kn (2.0), GA3 (1.0)
Callus embryogenesis, complete plantlet
formation Mehra and Saroj, 1979
Golden Crown Lateral buds MS -- Multiple shoots Chen et al. , 1980
M.27 Leaves MS -- Adventitious shoots Constantine et al., 1980
Malus pumila Apical buds MS -- Multiple shoots Haung and Millikan, 1980
Golden Delicious, Co-op series 17, M.2, M.13, M.16, MM.109 and MM.110
Shoots tips, Axillary buds
MS BA, GA, Kn, IBA Shoot proliferation roots Lundergan and Janick, 1980
Golden Delicious Seeds MS BA, GA, Kn, IBA Embryogenesis, complete
plantlets Mehra and Sachdeva,1880
Golden Delicious, Griffith Shoot meristem MS and MS + B5 vitamins -- Micrografting Shu Ching and Millan, 1980
Malling Merton root stocks- M.104, MM.106 and M.109
Lateral buds QM, with vitamins of WS BA, PG, IBA Proliferation
No response to PG Snir and Erez, 1980
M.7 Shoot tip MS (1/2) BA (2.2), IBA (1-3) Axillary buds and roots Werner and Boe, 1980
Supreme Red Delicious, Wellspur Delicious
Shoot tip MS BA Etiolated plants Anderson, 1981
Malus pumila Mill. In vitro raised plants -- -- Acclimatization of
plantlets Brainerd and Fuchigami, 1981
Delicious Anthers MS(1/2) BA, IBA, GA3 Plantlet formation Fei and Xue, 1981
MK, M.25,M.26, MS,JG, andCOP
Shoot tips MS MS+BA Lateral shoots, complete
plantlets Gayner et al., 1981
M.9 Shoot tips LS BA (2.0), IBA (0.1), PG Rooting James and Thurbon, 1981
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Golden Delicious Cotyledon, hypocotyl, leaf
MS + B5 vitamins BA, NAA Callus, embryoids and
complete plantlets Liu et al., 1981
M.26 Basal callus MS -- Shoot regeneration Nasir and Miles, 1981
M.26,M.27,MM.104 and Stark Spur
Shoot tips MS 2-chloro3-phenyl – propionitrile
Rooting Nemeth, 1981
Granny Smith Axillary buds MS & MS(1/2) lq IBA (10) NOA (10) Rooting Sri skandarajah and Mullins, 1981
Malus asiatica and Malus prunifolia
Shoot tips MS BA, IBA Differentiation of plantlets Wang, 1981
A2 Buds MS PG, IBA Rooting Welender and Huntrieser, 1981
Delicious Anthers MS(1/2) BA, IBA, GA3 Haploid plants Xue et al ., 1981
M.26, Nothern spy, Summer Rambo, Nugget ,Ozark Gold, Spartin
Shoot tips MS, LS, (lq) PG, BA (1.0), IBA(0.1), GA3(0.5)
Proliferation, rooting Zimmerman and Broome, 1981
M.7, M.26, MM.106 Shoot tips MS BA (1.0), IBA (1.0), GA3 (0.1) Axillary bud, rooting Joung, 1982
BA, IBA, GA3 Lane, 1982
BA, IBA, GA3 Lane and McDouglad, 1982
Almey crab apple Shoot tips MS BA (2.0) Axillary bud, rooting Singha, 1982 a
Crab apples Shoot tips MS BA (1.0), NAA (0.2) Increase in Agar conc.
decreases shoot proliferation
Singha, 1982 b
M.27, M.9, M.26, MM.111, Mc Spur, Jonathan, Delicious
Shoot tips MS BA Rooting Sriskandarajah et al ., 1982
Jonathan Shoot tips MS GA3 Adventitious roots Takeno et al ., 1982
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Crab apples Shoot tips MS NAA, BA Proliferation, rooting Wangstreet, 1982
Delicious Shoot tips MS, LS, J, CH BAP (1.0), IBA (0.2) Proliferation rooting, callus organogenesis
Cheema and Sharma,1983
Not mentioned Stigma, style, ovule MS –– In vitro pollination, callus Globins, 1983
M.9 and M.26 Shoot tips LS BA (2.0), IBA (0.1), PG, Adv. Roots James, 1983
Golden Delicious Cotyledon, hypocotyl, leaf
MS + B5 vitamins BA, NAA Callus, embryoids and
complete plantlets Liu et al., 1983a, b
Malus pumila, Top red, Irradiated Anther MS, Millar, 2,4-D, IAA, Kn Haploid plantlets through
callus Lespinasse et al ., 1983
Orei Haploid callus 8P (Kao and Michayluk, 1975) and Modified MS
BA, IAA Callus Niizeki et al ., 1983
M.26 Shoot tips MS BA, IBA Complete plantlet
formation, acclimatization Welender, 1983
Not mentioned Lateral active and dormant buds
Lapoivre macronutrients, MS micronutrients, walkey- vitamins
BA, IBA, IAA Complete plantlets Broome & Zimmerman, 1984
M.7 rootstock Shoot MS(1962) without Glycine BA(0.82) Complete plantlets, acclimatized in field
Dunstan and Turner,1984
M.25 Micropropagated plantlets and callus
MS + Ammonium salt of
BA (1.0), GA3(0.1), NAA (1.0) Rooting of shoots,
organogenesis Jones et al ., 1984
Red Delicious Cotyledon Nitsch + Malt extract BA (4.0), IAA (0.2), 2,4-D (4.0)
Adv. Shoots and roots Kouider et al ., 1984 (a&,b)
Jonathan In vitro grown leaves B5 and MS 2,4-D (0.8), BA (0.5) Embryiods, roots Kouider et al ., 1984 (c)
Phiriki,Golden Delicious Seeds, cotyledons MS BA, IAA, 2,4-D, NAA Embryogenesis Organogenesis,
rhizogenesis
Rubos, 1984 Rubos and Pryke, 1984
Delicious, Golden Delicious Anthers, pollen grains MS, N6 Kn (0.93), IAA (22), 2,4-D (1.8) Complete plantlets Xue and Niu, 1984
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Mutshu, Northern Spy, Nugget, Ozark Gold, Rome Beauty, Spartan, Spruce Rome, Stayman, York
Imperial, Summer Rambo
Shoot tip meristem Quorin’s (1977) macronutrients, MS(1962) micronutrients, Walkey(1972) vitamins
IAA, IBA(1.47) Complete plantlets Zimmerman(a ),1984
Summer Rambo, Nugget, Ozark Gold, Spartin
Shoot apices Lateral buds
MS BA, IBA Complete plantlets acclimatization
Zimmerman (b), 1984
Akero Callus from shoot segments
MS + MS(1/2) BA(1-5), IBA (low) Differentiation Evaldson, 1985
Delicious and
York Imperial Shoot apices MS(1/2) IBA (0.33) Rooting Evaldson, 1985
M.26 Shoot apices Lapoiver (½) IBA (0.3) Rooting Lee, 1985 (a&b)
Jonathan Embryos, cotyledons Nitsch’s salts (1969) BA Shoots Kouider et al ., 1985
Malus bacata and Malus spectasbilis, M.4,Fuji
Shoot apices MS(1/2), LS, CH BA (2.2-8.8), IAA, IBA Complete plantlets, In vitro micrografts
Wang et al, 1985
Delicious and
York Imperial Shoot tips MS(1/2) IBA Rooting Standardi, 1985
Delicious Shoot tips MS + MS(1/2) BA, IBA Rooting Zimmerson, 1985a
Summer Rambo, Nugget, Ozark Gold, Spartin
Shoot tips MS + MS(1/2) BA, IBA Rooting Zimmerson 1985b
M.26, Nothern spy, Summer Rambo, Nugget ,Ozark Gold,
Spartin
Shoot apices MS IBA (1.5) Complete plantlets Zimmerson &Fordham, 1985
MM.126 Shoot apices MS(macro& micro)+B5(vitamins)
BA (2.0) Hardiness in shoot cultures
Caswell et al, 1986
M.25, M.27, M.106, M.9 and Jork
Shoot apices MS BA Multiple shoot formastion
Collet and Le 1986
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Root stock A2, M. Sylvestris (L) & M. Domestica (Borkh)
Shoot tips MS BAP (4.4μM)
Lateral shoots at base Nordstrom & Eliasson, 1986
Red Delicious, Jonathan Embryo, shoot tips MS + CW, Clorox(10-20%) NAA (0.2), BA (1.0) Complete plantlets Skirvin et al, 1986
Not mentioned Shoot apices MS TDZ Shoot proliferation Van Niubreek et al 1986
Fuji Shoot apices MS, MS(1/2) BA (1.0), IBA(2.0), GA3(0.1)
Shoot proliferation, Rooting
Yoe et al, 1986
M26 Leaf mesophyll, cotyledons and
zygotic embryos
Gamborg (B5) 2,4-D, IBA, NAA, BA Shoots Barbieri and Morini, 1987
Empire, McIntosh, Delicious, Triple, Red Delicious, Vermont Spur Delicious
Shoot tips LS BA(4.4), IBA(0.5), GA3(1.3)
Multiple shoot formation
Byeong et al., 1987
M.25, M.26, M.27, MM.106, M.9, Jork
type and Cyadona oblonga (Cyadon apple)
Proliferated
shoots MS IAA Rooting Collet and Le, 1987 (a&b)
Antanovo, Obykno, Vennaya, Jonathan,
Severnyl sinap, Shafranyl and Wealthy pippin
Stem segments, shoot tips, anthers and leaf discs
MS BA Callus Gladyshava, 1987
KSC.3 Shoot tips MS IBA Rooting Hicks, 1987
Jonathan, Prairie fire, Crab apple
Cotyledons, Embryos MS, Myoinositol(100), Ascorbic acid(50)
BA(2.0), NAA(0.1) Adventitious and axillary shoots (for disease screening)
Joung et al., 1987(a)
Jonathan, Prairie fire, Crab apple
Shoot tips MS BA(4.0), IBA(0.2), Multiple shoot proliferation
Joung et al., 1987(b)
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Many Micropropagated plants
-- -- Greater field performance
Karnatz, 1987
Antanovka and many
others Shoot cuttings MS BA Callus
Kudryarkin and
Evseena,1987
Malus × domestica. Callus, Irradiated pollen, Endosperm
MS γ-Rays, BA Abnormal embryos Nicoll et al., 1987
Malus spp. In vitro grown plants
-- -- Observed field performance
Rosati and Gaggioli, 1987
Top Red, Starling Anther MS BA, IBA, NAA Callus, somatic embryogenesis
Zhang et al., 1987
Green Sleeves Mesophyll KSMP -- Callus Droughty and Power, 1988
Not mentioned Leaf tissue MS BA(2.0), NAA(0.5) Shoots, organogenesis James et al., 1988
Graf Uhlhorus, Augustthalvill
Sprouts from winter dormant
cuttings
MS BA and IBA in 6:1 ratio Shoot proliferation Lamier, et al., 1988
Malus prunifolia strain MO84
Shoot tips, single cells, in vitro grown roots
LS BA, IBA Axillary shoots and roots
Masuda et al., 1988 a, b
M26, Greensleeves Leaf discs MS BA(2.0), NAA(0.1) Shoot regeneration Ognjanov, 1988
Quince apple (Cyanodona oblonga)
Shoot tips MS, WPM IBA (1.0) Rooting Orlikowsko, 1988
M26 In vitro raised leaves
MS salts and LS vitamins,
containing BA (4.4) + NAA (0.5) Shoots
Predieri and Malavasi,
1988
Not mentioned Cotyledons, tigellum
MS IAA Callus, shoots Sinska, 1988
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Malus pumila, M. hupehensis, M. Zumi and M. rrobusta
Shoot tips MS, LS BA, NAA Resistance to fungal infections
Scheeve, 1988
M6 Coyledons, embryo
MS BA Shoots Stanis and Gyalvonauskis, 1988
Akero, McIntosh, McIntosh Wijick, Gravinstain, M26
Leaf segments, Stem segments
N6 BA(22.0), NAA(1.0) Shoots, roots Welander,1988
R1-49, Lodi and Erovan Unfertilized ovules and
embryos
MS BA, IBA, NAA Adventitious shoots Zhang and Lespinasse, 1988
M.26 Shoot tips MS IBA, Rooting Alvarez et al., 1989
Melrose Shoot tips MS NAA (0.001-0.1), BA (0.5-4.0) and GA3(0.1)
Shoots and roots Arello et al., 1989
Leaf segments Leaves with stomata Blanke and Belcher, 1989
M.9 Shoot tips MS, PG (162) BA (2.0), IBA (0.1) Adventitious shoots,
roots Christine et al ., 1989
Jonagold and root stock M.26)
Shoot tips MS BA (0.1) Echard, 1989
Several cultivars Leaf discs N6, LS BA, TDZ, NAA Shoots Fasiola et al., 1989
MAK.9, M.26, MM106 Shoot segments MS BA (0.5-1.0) Axillary morphogenesis
Ivanova, 1989
Not mentioned Shoot tips, seedlings
MS BA Shoot proliferation Lankes, 1989
Many In vitro grown leaves
MS BA Irregular stomata Blanke and Belcher, 1989
Five cultivars Lateral buds MS IBA (2.4), BA (2.2) and GA3(0.3)
Axillary shoot proliferation
Petrovic, 1989
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M.9 Shoots MS Different Shoot rejuvenation Webster and Jones, 1989a
Delicious, Triple Delicious,
Red Chief Delicious and Starkspur Delicious
Shoot tips,
axillary buds MS BA+ IBA+PG Shoot proliferation
Zimmerman and Fordham,
1989
Not mentioned Leaf & fruit tissue
MS and LS -- Protoplasts, callus Anderson, 1990
M.7 Shoot tips MS IBA (1.0-3.0) Rooting Correa et al., 1990
Different Leaf segments MS BA Shoots Hedtrich Hedtrich, 1990
M.26 Shoot tips MS BA (2.0), NAA (0.5) Proliferation, multiplication
Lankes, 1990
Mark and Gala Shoot tips MS -- Rooting Lankes and Zimmerman,
1990
M.26 Shoot tips MS BA (2.0), NAA (0.5) Shoot multiplication Lee et al ., 1990
Gal, Royal Gala and Jonagold
Shoot tips stem sections
MS with variable ammonium nitrate concentration
BA, IBA Adventitious roots Sriskandarajah et al., 1990.
Mark Leaf discs MS and N6 BA + TDZ Shoot regeneration Theiler and Theiler, 1990
American, Summer Permian, Golden Delicious, Fuji, Ralls and Jonathan
Shoot tips
MS (1/2), LS Casein Hydrolysate (CH-300-500)
BA (0.5–1.0) Shoot and root formation
Wang, 1990
American, Jonathan and Summer Permian
Anther MS(1/2) GA (0.06), IBA (0.5), BA (1.0) and IAA (1.5)
Shoot and root formation
Xue et al., 1990
Delicious, Ralls, Rainer and Golden Delicious
Anther MS(1/2) 2,4-D (0.4), Kn (0.2) and IAA (3.8)
Embryo induction Xue et al., 1990
Root stock M.111 Shoot tips MS BAP, NAA Rooted shoots Arello et al., 1991
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Golden Delicious
Inter-nodal segments of in vitro grown shoots
MS BA(4.4), TIBA(1.0) Complete plantlets Belaizi et al., 1991
Golden Delicious Shoot tips MS + Potassium humate (50) IBA, KH Rooting Bhraldi et al ., 1991
Royal Gala and M.26 Leaf segments MS TDZ, BA , Kn Z(Zeatin) Mteatopolin(TOP)
Multiple shoots Dobranski et al., 1991
Not mentioned Callus, axillary buds
MS Blue light Pigment cyanidine-3-galactoside
Komiya, 1991
Not mentioned Shoot tips MS BA(1.0) +8HQS Shoot proliferation Machado et al., 1991
P60 Polish Clonal Apple Stock
Shoot tips MS+ L-arginine(200), L-proline(200)
PG(80) Rooting Orlikowska, 1991
B9, Otttawa, P2 and P22 Shoot apices MS PG + BA Multiple shoots Webster et al., 1991
M.9 and York Shoot apices MS PG(162), GA3(1.0)and IBA(2.0)
Rooting Caboni et al ., 1992
Starkrimson, Bisbee, Ranier, Qiujin and Liaofu
Leaf mesophyll, protoplasts, ovules
MS BA Callus, caulogenesis Ding and Chao, 1992
Royal Gala and apple
rootstock 'M.26' Leaf discs MS TDZ, BA, TOP, ZEA, KIN Shoots Dobranski, et al., 1992
Gala Leaf discs, embryos and cotyledons
MS +B5 vitamins BA(4.0), NAA(1.0) Caulogenesis and Rhizogenesis
Gerson, 1992
Jork, Nicolai, Elester and Golden Delicious
Micro-cuttings MS BA + IBA Complete plantlets Klerk and Brugge, 1992
Jork9 Shoot tips MS + Sorbitol (0.167M) BA Shoot proliferation Moncousin et al ., 1992
Jonathan Shoot segments MS IBA, ABA Axillary shoot proliferation
Noiton et al., 1992
Ph.D. Thesis of Wilayat Rizvi E.mail_ [email protected]
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M9, Jork Stem segments MS + B5 vitamins BA, GA3 and IBA Rooting Pawlicki and Welender, 1992
Ottawa3 Shoot tips MS and B5 BA, IBA Rooting Puente and Marin, 1992
Many Seeds, cotyledons
MS BA Caulogenesis Stanys et al ., 1992
Finnish root stock YP Shoot tips and split shoots
MS KIBA Rooting Uosukaimen, 1992
Ottawa 3(M. baccata x M. sylvestris)
Leaf segments MS, B5 and N6 Gelrite BA(1.0), NAA(1.0) Adventitious shoot proliferation
Welender and Maheswarn, 1992
Gala Shoot tips MS IBA (1.5) Root primordia Harbage et al., 1993
Not mentioned Protoplasts from young leaves
MS + B5 vitamins BA + NAA + 2,4-D Complete plantlets Huancaruna and Schieler, 1993
Marubakaido (M. prunifolia) Wild Borkh and Megumi (M. domestica)
Shoot apices MS BA(2.0), GA3(1.0) Shoot proliferation Schuch and Peters, 1993
Delicious Transformed shoots
MS BA Genetic transformation
Sriskandarajah, et al., 1993
Gala Shoot tips MS (1/2) IBA(0.15, 1.5, 15 and 150) Rooting Stimart and Herbage, 1993
M.26 Shoot tips MS NAA/ Agrobacterium Rooting Sutter and Luza, 1993
M9 and Jork Stem segments MS IBA Cambium and
root primordial
Welander and
Pawlicki, 1993
Malus pumila In vitro grown Leaves
MS BA Shoot Durham et al., 1994
Golden Delicious Shoot apices MS + B5 vitamins BA(1.0) + PG Shoot proliferation Modgil et al ., 1994
Delicious Anther MS BA Complete plantlets Niu et al., 1994
Katja, Summer Red Zygotic Embryo MS GA3 Complete plantlets Raen 1994
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Empire, Freedom, Liberty,
Golden Delicious,
McIntosh, and Mutsu, M.7A
and M.26
Shoot apices, leaves
N6 , MS BA(22.2, 31.1), Antioxidants, antibiotics
Shoot proliferation Yapes and Aldwinekle, 1994(a,b)
Senshu, Gala and Fuji Leaf segments MS BA(2.0), NAA (0.2) Leaf callus and
plantlets Dong et al., 1995
Starkrimson, Raneir, Qiujin and Liaofu
Cotyledons, protoplasts
MS BA, IBA, IAA Callus Ding et al., 1995.
40 cultivars Anther, pollen MS+ Charcoal BA, IBA Complete plantlets Hidano et al., 1995
Six cultivars and M. zumi
Anther MS 2,4-D(0.2-1.0), NAA(0.2) Somatic embryos Hofer and Lespinasse, 1995
Dwarf apple cultivars Shoot apices MS BA,GA3
Vitrification of shoots by increase in BA conc. but decrease in GA3conc.
Niu et al., 1995
Aroma and others Seeds, Nucellus and endosperm
MS 2, 4_D(1.0), , BA(1.0) Somatic embryos Wallin et al., 1995
Starcrimson, Rainer, Quijin and Liafo
Leaf segments MS etc BA (5-10mg/l) and NAA (0.2ng/l)
Shoots Aping and Fang, 1996
Gloster 6900 Cotyledons MS NAA(5.0), BA(0.25), TDZ Secondary somatic embryogenesis
Daigny et al., 1996
Starkrimson, Raneir, Qiujin and Liaofu
Leaf segments MS BA(5.0), NAA(0.2) Adventitious buds Ding and Wang,1996
Gala Leaf segments MS BA(1.0), NAA(0.5), 2,4-D(0.5)
Direct somatic embryogenesis
Dong et al ., 1996
M.26 Shoot apices MS BA(low), IBA(low) Caulogenesis Ferradini et al., 1996
Fuji Shoot apices MS 2000-3000Lux for 10
BA (4.44) , IBA (0.5) and casein hydrolysate
Shoot proliferation He Shu Tao et al., 1996
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hour/day (200mg/l).
Six cultivars and M. zumi
Anther MS 2,4-D(0.2-1.0), NAA(0.2) Complete haploid plantlets
Hofer and Lespinasse, 1996
Gala and FTRI Leaf segments, internodes and petioles
MS BA(1.0), IBA(0.1) Adventitious shoots Hyae et al ., 1996
Polish dwarfing root stock P-59
Shoots MS, MS(1/2), WPM Agar (0.6%)
IBA(1.0) Roots Usek, 1996
MM.106 In vitro raised plants
-- --
Field performance
Root hairs disappeared, less stomatal index
Vegvari, 1996
M.9 Zygotic embryos MS BA(2.0), GA3 (2.0) Multiple shoots Wang et al ., 1996
Nezalezhuist, Idared and Florina
Leaf segments MS(1/3) BA(5.0), NAA (1.5) Shoots Bartish and Korkhovoi, 1997
Compact Spartin Shoot tips MS + Ca(NO3)2 -- Rooting Drought, 1997
Not mentioned Stem slices MS -- Adventitious roots Jasik and Klerk, 1997
Fuji Leaf segments MS+CH (100 and 200) TDZ(2.0) Adventitious shoots Hyae et al ., 1997
McIntosh Stem sections MS -- Multiple shoots Karhu, 1997
Jork9 Shoot tips MS Ethanol Growth inhibition Klerk et al ., 1997
Not mentioned Shoot tips MS with Hydrolysed agar from Difco
BA, IBA Controlled hyperacidity in medium
Marga et al ., 1997
M.26 Uninodal stem segments as synthetic seeds
MS IBA (24.6 µM )and sucrose (15 g 1−1)
Complete plantlets Piccioni,1997
Not mentioned In vitro raised
micro-cuttings MS Different Rooting Puntae and Martin, 1997
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Malus pumila Shoot tips MS Different Control over oxidative browning
Kumar and, Kumar, 1998
Malus domestica cv. Gala and Royal Gala
Shoot tips and axillary buds
MS Different
Field assessment of
in vitro raised plantlets for somaclonal variation
McMeans et al ., 1998
Red Delicious and
Amarican
Shoot tips, axillary buds, nodal stem segments, leaf
segments, root segments, seeds and cotyledons
MS, White, WPM BA, Kn, NAA, IAA, IBA, 2,4-D
Direct Multiple shoot induction and rooting
Bhat et al. , 1999
Ambri and Golden Delicious
Shoot tips, axillary buds, nodal stem segments, leaf
segments, root segments, seeds and cotyledons
MS, White, WPM BA, Kn, NAA, IAA, IBA, 2,4-D
Direct Multiple shoot induction and rooting
and callus formation
Rizvi et al. , 1999
Fuji, Junagold, Ringo and M9
Meristem derived callus
MS(1962) TDZ(0.2), IAA(0-1), ABA(0-1)
Complete plantlets Saito and Suzuki., 1999
Gala, Royal Gala & Jonagold
Different MS manipulated with variable NH4NO3 and KNO3,
Different Rooting SriSkandarajah et al ., 1999
M.26, Gravenstain Shoot tips -- -- Field establishment Zhul et al ., 1999
Jork9, M.26, Gala
and McIntosh
vegetative
shoot apices
MS medium without
glycine
17.8 7M BA, 2.7 7M
NAA and 250mg l.0m1
cefotaxime
Complete plantlets Caboni et al., 2000
Moscatella and Starkspur Red
Single node shoots
Four media -- High shoot proliferation
Negri et al., 2000
Ph.D. Thesis of Wilayat Rizvi E.mail_ [email protected]
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Greensleeves Stem sections MS GUS-gene from Brassica napus
Transgenic apple with high load bearing capacity
Gittins et al ., 2001
'Marubakaido' and 'M.26' apple
Apical shoots MS(1/2) + vermiculite and Plantmax
IBA Complete plantlets Hoffmann et al., 2001
Marubakaido & 'M.26 Apical shoots MS (1/2) + vermiculite and Plantmax in place of agar
IBA Rooting Lambert & Tepfer, 2001
Transgenic apple Malus pumila Mill.
Vegetative tissues
MS Different Gene expression Stefano Predieri, 2001
Marubakaido and M.26 In vitro raised shoots
MS(1/2) IBA, Adventitious root induction
Hofmann et al., 2001
M.7 Leaf segments MS BA, CPPU Callus Martins et al ., 2001
M.26 Leaf tissues MS Encapsulation materials Synthetic seeds Sicurani et al., 2001
M.26 Uninodal in vitro raised shoots
MS Encapsulation materials Synthetic seeds Brischia et al., 2002
Royal Gala, M.26 Leaf explants Different (MS, WPM) TDZ, BA, TOP, Zeatin,Kn Shoot regeneration Dobránszki et al ., 2002
Six cultivars Anthers MS BA (4.4μM) + IBA (0.5μM) Shoots Kadota et al ., 2002
M.26 In vitro born Apical buds
PPM(Plant Preservative Mixture)
Sodium alginate Synthetic seeds Micheli et al., 2002
Different root stocks and cultivars of Malus
In vitro grown Scion and stocks
-- -- Allografts and autografts, complete
plantlets
Abreu et al., 2003
M.9 EMLA Shoots Semi solid and liquid MS BA, IBA Complete plantlets Chakrabarty et al., 2003
Malus xiaojinensis, M. pumila
Shoots MS BA, IBA Complete plantlets Cheng et al., 2003
Gala and McIntosh In vitro raised shoots
MS, SH and QL Agrobacterium rhizogenes infection at the base of
micro-cuttings in vitro
Rooting Damiano and Monticelli, 2003
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Jork.9 Shoots and stem segments
MS solidified and Liquid media with 10X vitamins
BA, IBA, GA3 Vigorous growth and proliferation
Damiano et al., 2003
Gala Shoot tips MS Different Slow growth for
storage Hao and Deng, 2003
M.26
Stem sections From
micropropagated shoots
MS
indole-3-butyric acid (IBA),
N,N -bis-(2,3-methylenedioxyphenyl)urea
and
N,N -bis-(3,4-methylenedioxyphenyl)urea
Rooting was enhanced when stem sections were treated
with IBA in association with MDOPU
Ricci et al., 2003
Queen Cox Shoot tips, leaf segments
DKW, MS with ½ NH4+ and
NO3-
PG + BA Adventitious shoot regeneration
Wilson and James, 2003
Malus domestica Borkh Anthers MS (modified) BA Embryoids, embryogenic shoots
Hofer, 2004
Ambri Seeds, In vitro raised Shoot tips
MS+ NH4+, NO3
- (30-60mM)
BA (1-10), TDZ (0-10), IAA(0.02), IBA(0.02)
Axillay shoots, roots Sharma et al., 2004
Transformed scion cv. ‘Florina’
Shoots tips, leaf tissues
-- rolB gene introduction Rooting Radchuk and Korkhovoy, 2005
MM.106 Leaf segments MS BA + NAA Adventitious shoots, Modgil et al., 2005
Fuji, Gala -- -- M aminoethoxyvinylglycine (AVG)
Shoot regeneration, genetic transformation
Seong et al., 2005
Braeburn Anthers MS Different Complete plantlets Vanwynsberghe, 2005
Malus pumila Fruit tissue MS liquid Different Oxidative activity Allan et al., 2006
M.9 Shoot tips MS BA(0.5), IBA(0.1),
Kn(0.47)
Complete plantlets with 80% survival rate
Dalal et al., 2006
Malus domestica Leaf mesophyll MS Cell wall digesting enzymes, BA, IBA
Transformed disease resistant plants
Dandekar et al., 2006
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Malus prunifolia (v. Marubakaido) and M. pumila (cv. M9)
Immature embryos (40 days old) obtained after
cross pollination
MS(1962) + Caesin hydrolysate (500)
GA3(0.5), IAA(2.45), Kn(1.0)
Embryo rescue and new seedlings
Dantas et al., 2006
Apple x Japanese pear hybrid
Ovules, immature embryos
MS BA, Shoots Goani et al., 2006
Orin Callus from fruit tissue
callus proliferation medium (CPM) containing MS salt NN organic component
BA (1µM), 2,4-D(4.5µM) Callus growth but no morphogenesis
Liu et al., 2006
Schneid Anther Modified 8P BA, cellulose, pectolyase Protoplasts Niizek et al., 2006
M.9 Nodal sections with two lateral buds
MS (with 1/3 Nitrogen source) BA (0.8) Low photosynthetic
rate in shoots Zanandrea et al., 2006
Golden Delicious Shoot tips MS supplemented with 1 mg l-1 each of thiamine,
pyridoxine, niacin BA, GA3
Reduction in browning and
multiple shoots
Manha, N. and Motabllebi 2007
Ambri
Shoot tips MS BA, NAA, IAA, IBA,2,4-D, PG
Direct Multiple shoot induction and rooting and callus formation
Rizvi et al. , 2007
M. sieboldii × M. domestica sp
In vitro grown Scion and stocks
MS BA, IBA Allografts resistant to Phytoplasma
Bisogenin et al., 2008
Malus sieboldii Actively growing shoots
MS, QL, WPM and DKW.
BA (4.44 μM), IBA(0.25μM) and GA3 (0.28μM)
Complete plantlets, Hardening with survival rates of 90–100%.
Ciccotti, et al., 2008
Malus domestica Borkh Anthers MS Different Androgenic haploids and diploids through andro-embryogenesis.
Hofer, 2008
Malus domestica Borkh Shoots MS with MES (morpholino 2.0 mg/l TDZ, with 0.2 mg/l Complete transgenic Nabeela, et al., 2009
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Golden Delicious and ‘MM111’ rootstoc
ethanesulfonic acid) NAA plantlets with antifungal genes
Maharaji and Chambura
Shoot tips MS BA, TDZ, PG, IBA
Direct Multiple shoot
induction and rooting and callus formation
Rizvi et al. , 2009
Malus domestica Borkh
X Malus robusta
G.935 -- --
Complete transgenic plantlets with resistance to fire blight and crown rot
Telias, 2009
Malus hupensis (Pamp) Seedlings MS BA (2.22μM), NAA(0.5μM)
Complete transgenic
plantlets with resistance to different pathogens
Zhang et al., 2010
Malus domestica Borkh Florina, Romus3, Romus4, Colmar, Rebra, Goldrush, Idared
Nodal Segments
MS - msupplemented with Lee and Fossard (LF) [20] vitamins,
BA (2.22μM), NAA(0.5μM) Butiuc-Keul et al., 2010
Malus domestica Borkh -- MS - Different
Complete transgenic plantlets with resistance to different pathogens
Bhatti and Jha, 2010
Malus pumila MM.106
Shoot tips MS medium IBA (0.4 µM) BA (4.43 µM) Complete plantlets Bahmani, et al., 2010
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responsible for proliferation of dormant buds. Elliot (1972) working
independently observed leafy shoot formation from shoot apices of Granny
Smith variety cultured in vitro when supplied with BA (0.23 mg/l) or Zeatin
10.02mg/l). In the same year Chong and Taper (1972) observed callus
initiation from apple shoot apices grown in presence of I sorbitol (D-glycitol)
as a carbon source. This was followed by another report by Dutcher and Powel
(1972) who noticed multiple shoot formation when BA (0.1-1.0 mg/l) was
added to medium. Complete plantlet formation from shoot apices of five root
stocks without any proliferation was observed by Quoirin (1974). Studies
related to the effect of light intensity on growth and chlorophyll content and
sorbitol metabolism of Malus shoots grown in vitro were carried out by Chong
and Taper (1974). Again in 1974 Walkey observed positive impact of
polyvinyl pyrolidone (PVP) in liquid medium for 4-8 weeks during in vitro
culture of apple shoot apices to eliminate the inhibitory effect of oxidized
phenolic compounds on meristem tips. Jones (1976) culutred shoot apices of
M.7 and M.26 root stocks on Quoirin’s medium (1974) with vitamins used by
Wetmore and Sorokin (1955) supplemented with floridzin and phloroglucinol
(10-3
M), BA (0.5 mg/l), IBA (1mg/l) and IAA (1mg/l) and reported that
phlorizin and phloroglucinol promote the growth of apple shoots with
pronounced effect. Continued efforts in this direction by Jones and Hatfield
(1976) showed that phloroglucinol with auxin doubled the number of M.7
shoots which later on produced adventitious roots. Phloroglucinol, Caffeic acid
and Catechol were found ineffective. The most effective auxin treatment was
IBA (5.0 µM). Jones et al. (1977) published first complete protocol for large
scale micropropagation of apple.
Abbott and Whitely (1976) worked on meristem tips of seedling and
adult trees of Cox’s Orange Pippin grown in green house and reported
proliferation of mass shoots followed by their individual rooting on LS
medium. Explants from adult trees showed only axillary bud proliferation bud
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seedling explants showed proliferation of axillary as well as apical buds.
Hopgood and Ferrel (1977) cultured shoot apices of M.26 root stock on MS
medium supplemented with BA and reported axillary shoot proliferation. It
was followed by a report published by Huth (1978) who observed that BA
(8.9µM) and NAA (1.1µM) were best for the establishment of very small
(8.9mm) ‘Jonathen’ meristem tips and reduced NAA concentration (0.3µM)
favoured optimal shoot growth. Rooting (70%) was achieved using NAA (5.4
µM) and results improved with GA3 (0.3µM). To overcome difficulties in
acclimatizing the rooted shoots he developed a successful technique of grafting
the in vitro produced shoots onto apple seedlings in the green house. Lane
(1978) cultured meristem tips of active and dormant apical buds of Macspur
cultivar and reported reduced proliferation in presence of GA3 and NOA
whereas BA promoted it. Proliferated shoots were transferred to NAA based
medium for rooting.
Zimmerman (1978) reported more than 90% rooting from shoot apices
of Jonathan cultivar with IBA (0.5-1.5µM) or NAA (0.5-1.6µM). Other
varieties of apple were found to show better rooting on half-strength MS
medium with perlite-vermiculite (1:1) support instead of agar. Phloroglucinol
showed no influence on rooting. Wang and Sui (1978) made preliminary
studies on in vitro produced stock plantlets from their growing points of buds
and reported their usefulness in apple micropropagation. Work on grafting of
in vitro produced apple shoot apices onto decapitated root stock seedlings was
carried out by Alskief and Villmur (1978) but their attempts were not
successful.
Mehra and Schdeva (1979) raised callus from shoot apices and stem
segments of Golden Delicious cv. on White’s medium (1943) supplemented
with NAA (4µM) + Kn (2ppm) + coconut water (Cw) (5%) + GA3 (1ppm)
which later differentiated into embryoids and normal leafy shoots in presence
of NAA (2ppm) + BA (2ppm) + Cw (15%) in two months. However,
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differentiation of complete plantlets with root and shoot system was not
observed by them. At the same time James and Thurbon (1979) cultured shoot
apices of M.9 apple root stock on Linsmaier-Skoog (1965) medium and
reported 60% rooting in presence on IBA (2.0 mg/l) and PG (162 mg/l).
Culture of shoot apices with their cut ends touching medium resulted in callus
formation at the cut ends. Best rooting had occurred when cuttings were placed
on medium containing IBA and PG for four days. The rate of rooting differed
between two lines of M.9 root stock and the differences persisted over several
subcultures. Continuous efforts in this direction lead Jones (1979) to culture
shoot apices of five scion cultivars (Malling Kent, Malling Sunitan, James
Grieve, Cox’s Orange Pippin and Golden Delicious) on MS medium
containing BA. The cultures multiplied about 5 fold every three weeks and
more than 80% shoots had produced roots. 90% plants survived after
transplantation.
Work on in vitro storage was initiated by Lundergan and Janick (1979)
who developed the technique of low temperature storage for in vitro raised
apple shoots. Results have however, shown varied effectiveness of cytokinins
in stimulation of the shoot proliferation of Golden Delicious cultures. Their
observations have shown that the most effective cytokinin was BA, the least
effective was 2iP while the impact of Kn was in between. The effect on shoot
elongation was, however, in reverse order. When BA (13.2-22µM) was added
to medium stunted shoots were produced. Normal growth was observed by
transferring the cultures to media with IBA (4.9µM) and BA or 2iP (4.4 µM).
Werner and Boe (1980) propagated in vitro M.7 root stock on half
strength MS medium augmented with BA (2.2µM). Rooting of shoots was
observed readily on one-third strength MS medium supplemented with IBA
(4.9-14.8 µM) and Agar (0.27%). Snir and Erez (1980) cultured shoot tip
sections of three cultivars (MM.104, MM.106 and M.109) in Quoirin’s
medium (1974) with vitamins used by Wetmore and Sorokin (1955) for four
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days on an orbital Shaker and observed increase in shoot proliferation rate.
They also observed inhibition in shoot proliferation by IBA. Wounding of cut
ends of shoots enhanced root formation and phloroglucinol showed no impact.
More published reports still continue to come in this direction.
Chen and Yang (1980) cultured branch apices of adult apple cv. Golden
Crown and obtained multiple shoots. Huang and Millikan (1980) obtained
similar results on apical buds. Lundergan (1980) reported impact of different
factors like phytohormones on apple shoot growth and proliferation. He
worked on Golden delicious, Co-op series 17, M.2, M.13, M.16, MM.109 and
MM.110 cultivars and reported proliferation of axillary buds on MS medium
containing BA, GA3 and IBA. Complete plantlet formation was observed. In
India Mehra and Schdeva (1980) succeeded in differentiating callus from
Golden Delicious apple shoots on BAP based medium. They also observed
embryo formation which subsequently developed into complete plantlets.
Zimmerman and Broome (1981) observed the favourable influence of
phloroglucinol for rooting in cultivar Spartin, with different concentrations of
IBA (0-4.9µM). Autoclaving did not alter the activity of PG for rooting.
However, PG reduced callus formation. In apple root stock A2, PG (1mM)
inhibited rooting of adult phase plant buds while PG (0.1µM) stimulated it in
presence of IBA (5µM). Both concentrations stimulated the rooting of the
shoots obtained from juvenile phase plants (Welender, 1981).
While working on several factors affecting rooting in vitro of
apple shoots, Nemeth (1981) tested synthetic auxins and reported that 2-chloro-
3(2,3-diclorophenyl) propionitrile was more effective than IBA in stimulating
rooting in M.26,M.27,MM.104 and Stark Spur. The best concentration reported
was 5µM. Sriskandarajah and Mullins (1981) worked on Granny Smith apple
shoots and induced 80% rooting in MS based liquid medium supplemented
with IBA (10µM) and NOA (10µM). However, 2,4-D was found inhibitory.
Same year James and Thurbon (1981) reported in vitro root and adv. shoot
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initiation from shoot apices of M.9 root stock and observed IBA (3mg/l) and
PG (1620 mg/l) to be most effective for inducing in vitro rooting.
Phloroglucinol acted as auxin synergist in the process. Multiplication of shoot
tips was found to be most favourable at BAP (1-2 mg/l) and IBA (0.1-0.5mg/l)
where as PG had no effect on shoot multiplication rate. Similar results on in
vitro rooting of shoot apices were published by Ancora (1981), Wang (1981),
Skirvin (1981), Gayner et al. (1981) and Werner (1980). Acclimatization of
aseptically cultured apple plants to low relative humidity was registered by
Brainerd and Fuchigami (1981). Sriskandarajah et al. (1982) reported that
cuttings made from proliferating cultures of Jonathen and Delicious grown with
continuous illumination (90-100 µEm-2
S-1
with cool white fluorescent tubes) at
26oC rooted better than those from cultures grown on 16 hour photoperiods
(20-30 µEm-2
Sec-1
) at 23
oC. Rooting percentage and number of roots increased
with increasing number of subcultures.
A study was carried out on the influence of Agar concentration on in
vitro shoot proliferation of crab apple cv Almey and it was reported that
greatest shoot proliferation at 0.3% or lower agar concentration occurred on the
MS medium. Increase in agar concentration resulted in decrease in shoot
proliferation and growth (Singha, 1982). He also successfully produced
plantlets from Almey crab apple shoot apices on MS medium with BA (0.2
mg/l). In the same year, Wangstreet (1982) observed proliferation of axillary
shoots from the apices of crab apple on MS medium augmented with BA and
NAA. The shoots produced roots after subculturing and the complete plantlets
formed were later transplanted to the soil. Cheema and Sharma (1983)
observed multiple shoot formation in several apple cultivars. Half strength MS
medium favoured the development of highly hydrated shoots, which were
sensitive to injury and initiated much basal callusing. Excellent growth of shoot
apices was supported by MS medium fortified with BA (1.0mg/l) + IBA (0.2
mg/l). Reports on adventitious root formation in vitro from shoot apices of M.9
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and M.26 root stocks on MS medium with BA (1-2 mg/l) came from James
(1983). IBA (0.1-0.5 mg/l) and PG (1620mg/l) favoured rooting. Welender
(1983) also worked on M.26 root stock shoot apices and successfully produced
complete plantlets. The plantlets were acclimatized in soil.
Reports on the protocol for effective in vitro studies on apple were
published by Zimmerman (1984). In his studies, he suggested use of calcium
hypochlorite as sterilant, modified liquid medium for establishment of explants,
half strength MS medium for rooting and soil mix (peat-like mix: vermiculite:
perlite,1:1:1) for acclimatization of in vitro grown plantlets. At almost same
time, Jones et al. (1984) cultured shoot apices of apple root stock M.25 and
observed callus formation which differentiated into complete plantlets on BA
based medium. Shoot segments of cv. Areko were cultured by Evaldson (1985)
on MS medium supplemented with IBA and BA which produced callus that
differentiated into plantlets. BA stimulated growth in association with low IBA
concentration but suppressed it in association with high IBA conc. Wang et al.
(1985) worked on micrografting and used aseptically cultured shoot tips as
scions. Plantlets thus produced were subjected to acclimatization.
Influence of temperature on in vitro initiation and development of apple
root stock M.26 has been observed by Le (1985). Shoot tip explants were
cultured on a modified MS medium supplemented with IBA (7.5µM). Root
formation was observed at 22±1oC when shoots were subcultured on half
strength Lepoivre medium (1977) with IBA (10µM) for a week and then
transferred to Lepoivre hormone free medium. Optimum temperature for in
vitro rooting was found to be 25oC. Impact of light, IBA and activated carbon
on Delicious and York Imperial cultivars was observed by Standardi (1985)
who reported proliferation of shoots on half-strength MS solid medium with or
without activated carbon for both root initiation and elongation. Good root
initiation was obtained following culture for a week in darkness with IBA (0.33
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mg/l) but without activated carbon. Root elongation was better in IBA free
medium. Presence of activated carbon had little effect.
Zimmerman (1985a&b) reported simplified method for rooting of apple
cultivars in vitro. Shoots obtained by proliferating apical buds initiated rooting
readily in darkness in a liquid medium containing sucrose (43.8mM) and IBA
(1.5µM). Increase in temperature during dark treatment from 25oC to 30
oC
improved rooting for several cultivars. Phloroglucinol did not show any
stimulating effect. Following this report, Caswel et al. (1986) observed
different ways by which in vitro apple shoot cultures developed hardiness.
Shoot cultures of MM.106 apple, subjected to a ten week short photoperiod,
low temperature hardening treatment, including a –3oC exposure followed by
5-7days at 20oC, were 4-8 times hardier than untreated shoot cultures. Cultures
grown on media with high sucrose concentration (3-14%) and not subjected to
hardening conditions had reduced shoot moisture content but hardiness up to 6
times. Van Nieuwbreak et al. (1986) reported stimulation of apple shoot
formation in vitro by the use of thidiazuran. Yoe et al. (1986) described, in
their work on shoot proliferation of Fuji apple, that shoots grew best on MS
medium containing BA (1.0mg/l), IBA (2mg/l) and GA3 (0.1mg/l). Reduction
in the strength of salts to half or one-fourth increased rooting whereas 16 hours
photoperiod increased toot number. Addition of activated charcoal to medium
resulted in the production of fewer but longer shoots. Some more reports
followed in 1986. Collect and Le (1986) observed in vitro multiplication of
apple (varieties-M.25,M.27, M.106, M.9 and Jork) and reported that the factors
affecting proliferation and growth rate of shoot apices differed among and
within the root stocks. Skirvin et al. (1987) also made similar observations and
recommended use of Clorox (10-20%) + surfactant (0.1%) for disinfection, MS
medium for culture establishment, BA for higher rate of shoot proliferation,
IBA in darkness for rooting and peat, perlite, sand mix (3:4:1) for
acclimatization of rooted apple shoots.
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In 1987, Gladyshava cultured shoot apices and stem segments of
different apple cultivars (Antonovska, Obykno, Vennaya Severnyl sinap,
Wealthy Pippin Shafranyl and Jonathan) in vitro and observed callus
formation. Collect and Le (1987) reported in M.26 apple cv. that minicuttings
taken from proliferated shoots were subjected to rooting. The best results were
achieved through brief pretreatment with IAA supplied at the cut end of the
cuttings. Adventitious rooting of microshoot cuttings of apple on MS medium
supplemented with IBA was reported by Hicks (1987). Byeong et al. (1987)
reported culture of different apple varieties (Empire, McIntosh, Delicious,
Triple Red Delicious and Vermont Spur Delicious) in vitro on Linsmaier and
Skoog (1965) medium supplemented with BA (4.4µM), IBA (0.5µM) and GA3
(1.3µM). All the apple varieties produced shoot with >1cm length in 24 hour
photoperiod. However, if the explant was placed horizontally on medium
number of shoots increased. A simple and effective method was explained for
screening shoot cultures of Malus for resistance to Gymnosporangium juniperi
Virginiae (Gjv) by culturing shoot tips of Jonathan Praire fire crab apple
cultivars in vitro with Gjv basidiospore suspension and evaluated for rust
symptoms (Joung et al., 1987). However, somaclonal variations could not be
obtained. Kudryarkin and Evseena (1987) observed the process of de-
differentiation in the shoot cuttings of one year old trees with or without cold
treatment in vitro in the period prior to over wintering. Callus formation
observed, in control, after 30 days, was 86.7-100% whereas in cold-treated
shoots no callus was formed except in Antonovka obknovennaya (77%).
Again in 1987, Karnatz working independently explained initial
development of self rooted in vitro grown apple shoot apices. Micropropagated
apple trees with their own roots displayed greater precocity of bearing than
conventionally propagated grafted plants. Rosati and Gaggioli (1987) analysed
field performance of micropropagated apple scion cultivars. It was followed by
a report describing the influence of photoperiod, apical meristem and explant
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orientation on axillary shoot proliferation of apple cultivars in vitro. Horizontal
orientation of explant with cut end slightly inside the medium and 16 hour
photoperiod favoured vigourous growth and better proliferation (Yoe et al.,
1987). Masuda et al. (1988) assessed relationship between abnormal shoot
formation and phytohormones. Abnormal shoots were produced on Linsmaier
and Skoog (LS) medium in presence of BA and this abnormal behaviour was
prevented by the addition of IBA (1-10 mg/l). Scheeve (1988) explained in
vitro selection among ideotypes with varying resistance to apple mildew. In
vitro cultures were established using the field resistance ideotype.
Influence of NAA and BA was evaluated by Arello et al. (1989) on the
in vitro multiplication of apple cv. Melrose. Multiple shoots (7-8) were
achieved using BA (2.0 mg/l), NAA (0.001mg/l) and GA3 (0.1mg/l) but the
shoot were of poor quality. Multiple shoots of superior quality were obtained in
presence of BA (0.5mg/l) + NAA (0.1mg/l) + GA3 (0.1mg/l). These shoots later
on rooted and were acclimatized to field conditions. Echard (1989) developed
an efficient in vitro cold storage method for shoot apices of apple (cvs.
Jonagold and root stock M.26) grown on MS medium + BA (0.1mg/L) at 2-
4oC. The apices were successfully preserved for 18 months at low temperature.
Growth of apices resumed after storage for 8 hours at low temperature
(15/10oC). In another report, Ivanova (1989) subjected shoot apices of cvs.
MAK.I, M.26 and MM.106 root stock for culturing on a two layer nutrient
medium (a liquid layer and a modified MS medium solidified with agar).
Axillary morphogenesis was 66-100% when BA (0.5, 0.7 or 1.0mg/l) was
added to liquid medium. The method proved two-fold efficient in comparison
to direct culturing on BA containing solid MS medium. Lankes (1989) tackled
with the problems related to in vitro propagation and multiplication of apples
with respect to age of tissue at the onset of culture, endogenous microbes,
phenols and vitrification. The promising techniques in future suggested by him
include: micrografting in which root stocks and grafts are cultured together
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(eliminating the need for root induction in the graft), tissue culture combined
with conventional techniques, eliminate of heat stable viruses and tissue culture
of own rooted apple culture. Rosati and Gaggiole (1989) evaluated orchard
response of micropropagated apple cultivars. He compared field performance
of tissue cultured (TC) apple trees with standard propagated (SP) apple trees.
TC trees were most vigorous and had same cumulative production (during
1981-86) as that of SP trees. However, TC trees lost juvenile character much
earlier in comparison to SP trees. Zimmerman and Fordham (1989) reported a
new technique to enhance axillary shoot proliferation. More axillary shoots
developed from subcultured apical shoots when placed inverted in the medium.
The effects were noticed in Delicious and its strains. Inversion of subcultured
shoots of Delicious reduced or eliminated tendency to early senescence.
Besides, axillary shoots rooted earlier in comparison to those oriented
vertically or horizontally.
In the apple root stock M.7 high rooting rate was reported from in vitro
grown shoot apices (Correa et al., 1990). Sprouts obtained in vitro were
cultured on MS medium containing IBA (0, 1.0, 2.0 and 3.0 mg/l). Best results
were obtained after 4 weeks on 75% strength MS salts containing IBA (10
mg/l). Lankes and Zimmerman (1990) observed impact of osmotic potential on
in vitro cultures of apple cvs. Gala and Mark root stocks and observed highest
percentage of roots when the medium with highest osmolality was used. Lee et
al. (1990) evaluated the impact of growth regulators and medium composition
on the growth of each stage in shoot tip culture of apple (M.26 cultivar). BA
(2.0mg/l) was found best for culture establishment, NAA (0.5mg/l) for callus
formation and BA (2.0mg/l) + NAA (0.5mg/l) for shoot multiplication. Best
results for rooting were obtained with NAA (0.1mg/l). Autoclaved charcoal
was found to reduce rooting percentage, root number but not root length.
Sucrose (30mg/l) and MS salts (25%) were best for rooting. Baraldi et al.
(1991) observed the impact of Potassium-humate (KH) on cv. Golden
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Delicious and reported its negative interaction with BA and thus enhanced
rooting. In presence of IBA, KH increased root number but reduced their
growth. Rooting was hastened with KH (50mg/l) and plants grew more rapidly
when transferred to soil. Komiya et al. (1991) also cultured shoot apices of
some apple cultivars and allowed them to produce callus which was placed
under blue light illumination to produce a colouring agent cyanidine-3-galacto-
side(I).
A new and efficient method for initiation and establishment of tissue
cultures of apple was proposed by Mechado et al. (1991) from adult material
where they reported that application of 8-hydroxyquinol sulphate (8HQS)
reduced contamination rate to a great extent and inhibited browning of explants
and media. Orlikowska (1991) proposed another efficient method of in vitro
propagation of shoot apices of apple plants grown in green house and observed
high proliferation rate of shoots. Rooting of shoots grown on medium
supplemented phloroglucinol (80mg/l), L-arginine (200mg/l) and proline
(200mg/l) was impaired by darkness during first five days. Webster et al.
(1991) reported varying degrees of success in micropropagation of some cold
hardy dwarf root stocks of apple shoot apices (cv.B9, Ottawa, P2 and P22).
Micropropagation was achieved readily in P22 and Ottawa. Improved shoot
production was reported with PG and cytokinin.
A study regarding root formation in microshoots of root stock M.9 Jork
on BA (2.0mg/l) in presence of light was carried out by Caboni et al. (1992).
Kelerk and Brugge (1992) extended this work and observed that delay in IBA
application decreased the percentage of rooting and also number of roots per
shoot. Rooting of shoots in continuous light without IBA was delayed
indicating that IBA was essential for maintaining the response. BA and IBA
were found essential for complete plantlet formation in cvs Jork, Nicolai, Elstar
and Golden Delicious and Cyadona oblonga. Moncousin et al. (1992), in his
studies, observed that shoot proliferation of microcuttings of Jork-9 was more
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successful on MS-medium containing Sorbitol (0.176M) than on medium
containing fructose (either added directly or through hydrolysis of sucrose).
Best results were obtained with sucrose (0.088M) during rooting although
Sorbitol (0.176M) used during both phases of culture gave similar results.
Welender and Maheswaran (1992) obtained multiple shoots from shoot apices
of cv. Ottawa-3 on MS and B5 media solidified with Gelrite. More reports in
this direction continued to get published. Puente and Marin (1992) reported that
if micro-shoots of root stock M.9 Jork were split 71.4% rooting was obtained in
contrast to 40% rooting by normal unsplit shoots. Most of the rooted shoots
(76.8%) obtained from split micro-shoots survived after planting in soil and
showed normal growth. Uosukaimen (1992) cultured shoot apices of Finnish
root YP and rooted them in media containing different sugars (glucose fructose
or sucrose) at 30g/l and KIBA (a potassium salt of IBA) at 25, 50 and 100mg/l.
Highest rooting was achieved in cultures maintained on proliferation medium
for 7 weeks when either sucrose or glucose was used in the rooting medium.
Shoots older than 7-weeks lost their ability to root. The optimum concentration
of KIBA was determined by the Concentration of sugar.
Shoot apices of Gala cv. when cultured on root inducing medium
containing IBA (1.5µM) + Sucrose (44mM) showed that some phloem
parenchyma cells became densely cytoplasmic within one day and formed
meristemoids which in turn gave rise to root primordial (Harbage et al., 1993).
The root primordia produced roots by 6th day. Stimart and Harbage (1993)
cultured shoot apices of Gala cv. of apple and then subcultured shoots (1.15 -
2.0cm long) on rooting medium containing IBA (0, 0.15, 1.5 or 150 µM) in
factorial composition with media at pH 5.5, 6.3 or 7.0. Root induction was
observed on half strength MS in darkness at 30oC after 4 days. Rooted shoots
were transferred to fine moist vermaculite after 18 days and then to soil after 57
days. Again in 1993, Schuch and Peters analysed the in vitro multiplication
potential of the shoot apices of apple (cvs. Marubakaido and Megume). Best
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results for shoot multiplication and growth were observed by him with BA
(2.0mg/l) with or without GA3 for Marubakaido and BA (3.5 mg/l) for
Megumi.
In Golden Delicious apple, Modgil et al. (1994) reported that
phloroglucinol added to the medium was ineffective but with Kn (1mg/l) and
BA (1mg/l) was highly effective for shoot proliferation. For culture
establishment MS medium supplemented with B5 vitamins, BA (0.5mg/l) GA3
(0.5mg/l) and ascorbic acid (200mg/l) has been suggested. Rooting in liquid
medium supplemented with sucrose (15mg/l) and BA (0.3mg/l) for seven days
has also been suggested. Yapes and Aldwinckle (1994) have reported
microprogation of 13 Malus cultivars and root-stocks. Shoot apices were
established using ascorbic acid, citric acid and anti-oxidants. The highest shoot
proliferation rate was obtained from root stock M.7A. They also observed that
cefotaxime (200mg/l) stimulated shoot growth and development but its higher
concentration (500mg/l) caused abnormal shoot formation.
Nui et al. (1995) studied the changes of endogenous hormones in dwarf
apple root stock during vitrification in vitro and reported that increase in BA
conc. and decrease in GA3 and IBA conc. caused vitrification of shoots. He
Shui Tao et al. (1996) worked on Fuji apple and reported highest growth and
proliferation of in vitro shoot apices under 2000-3000 Lux light intensity for 10
hour/day. The number of shoots was reduced by 26oC/22
oC and 24
oC/20
oC
day-night temperatures. The best medium for shoot proliferation and growth
was MS medium-supported with BA (1mg/l), IBA (0.1mg/l) and casein
hydrolysate (200mg/l).
The work on in vitro rooting of apple microshoots (cv. Polish dwarf root
stock P59) has shown that woody plant medium (WPM) with IBA (1mg/l) and
Shoei agar (6g/l) are best for achieving this goal. Rooting could further be
improved by adding proline (200 mg/l). Phloroglucinol had negative impact on
rooting (Usek, 1996). Drought (1997) worked on optimization of culture media
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for in vitro rooting of shoot apices of apple cv. Compact Spartin. He reported
that shoots raised in vitro did not root in single medium. Sucrose was essential
to achieve high root growth on agar but was not essential on watered
vermaculite Ca(N03)2 stimulated root initiation whereas long auxin treatment,
NH4NO3 and lack of substrate inhibited root emergence and growth. Karhu
(1997) in his work on microshoots of McIntosh grown on MS medium,
recorded their positive relationship with sucrose in shoot proliferation and
rhizogenesis. Puentae and Martin (1997), on the other hand, studied the rooting
potential of microcuttings (obtained from shoot apices) and reported that by
repeated subculture of shoots and splitting of their cut ends enhanced root
formation.
Klerk et al. (1997) studied the effect of ethanol and observed that in
mild concentration (0.2% v/v) it slightly stimulated the proliferation of shoot
apices through axillary branching in cv. Jork-9. Significant inhibition occurred
at concentrations of > 0.4% (v/v). Significant inhibition was also observed in
adventitious root formation at concs. of > 0.1% (v/v). The impact of ethanol
was found to be stage dependent.
Marga et al. (1997) worked on hyper-acidic effect in culture medium
during initial shoot tip culture of apple cvs. and suggested that it can be
controlled by using hydrolysed agar from Difco (0.7%). They also reported that only
oligosaccharides were found to reduce the occurrence of this developmental
abnormality during shoot proliferation. Later on, in 1998 Kumar and Kumar
suggested regular transfer of shoot explants in the fresh medium of same
composition for 2 to 3 days to check the problem of oxidative browning. Saito
and Suzuki (1999) have established the procedure for regeneration of complete
plants from meristem-derived suspension callus protoplasts obtained from
scion cultivars of apple (Fuji and Jonagold and Malus prunifolia var. Ringo
Asami Mo84-A). They suggest culture of the protoplasts on KM8P medium
containing IBA (2 mg/l), BA (1 mg/l), 2,4-D (0.4 mg/l), and MES (5mM).
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Caboni et al. (2000) have cultured shoot apices of Jork.9, M.26, Gala
and McIntosh cvs. in dark on glycine free MS medium supplemented with BA
(17.8µM), NAA (2.7µM) and cefotaxime (250 mg/l) for 20 days and then in
light so that callus produced at cut end redifferentiated into new shoots which
were rooted and finally established in soil. As far as the conservation aspect is
concerned, Negri et al. (2000) cultured shoot apices of single node shoot tips of two
apple cvs. Landrace (Moscatella) and Starkspur Red for four years. They suggest that
their technique of shoot tip culture in dark at 4oC for 18 months and longer can be
employed in making Germplasm Banks. Hoffmann et al. (2001) while working on
Marubakaido and M.26 cultivars of apple observed that use of Plantmax
instead of agar in MS medium favours efficient rooting of shoot apices during
acclimatization. Micheli et al. (2002) employed in vitro derived encapsulated
apical buds of M.26 rootstock in the formation of synthetic seeds. They suggest
double layer encapsulation for conserving apical buds as synthetic seeds. Hao
and Deng (2003) employed low temperature slow growth method for storing
shoot apices of Gala cv. of apple but observed that it resulted in a significant
change in DNA. Damiano and Monticelli, (2003) developed an improved
mechanism of rooting in micro-cuttings of Gala and MacIntosh cvs. of apple by
infecting their bases with Agrobacterium rhizogenes.
A study on the comparative impact of BA and TDZ (Thidiazuron) on the
shoot apices of in vitro raised seedlings of Ambri cv. of apple and showed that
TDZ is more effective than BA in terms of number of shoots formed and length
of each shoot Sharma et al. (2004). Recently, Radchuk and Korkhovoy (2005)
introduced rolB gene in Florina cv. of apple and then observed that rolB-
transformed plants produced altered root systems containing more fine roots
leading to significantly increased fresh root weight in the plant lines. In a very
recent report, Dalal et al. (2006) have reported forced shoot tip culture of M.9
root stock on MS supplemented with BA (2.22 µM), IBA (0.49µM) and Kn
(2.2µM). They reported 80% survival of shoots.
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II. Axillary Bud Culture
In vitro work on axillary buds of apple was initiated first by Pieniazek
and Jankiewiez (1966). They observed growth of dormant axillary buds
(collateral buds) in response to BA. Powel (1970) reported aseptic culture of
excised lateral buds of Cortland and Northern Spy cultivars of apple and
observed proliferation of axillary shoots with BA (0.1 –1.0mg/l). Dutcher and
Powel (1972) produced shoots from axillary buds of mature apple trees (cv.
Cortland) in stationary (MS) liquid culture media. The shoots had small leaves
and short internodes. Light and sucrose were found essential for growth. Low
ABA conc. prevented the growth of buds and 1% rooting of shoots was
observed in presence of IAA. However, IAA inhibited shoot formation. In the
same year, Walkey (1972) succeeded in regenerating complete plantlets from
axillary meristem tips of M.26 root stock on MS medium supplemented with
IAA (10mg/l). This work was followed by Quoirin (1974) who also reported
complete plantlet formation from axillary shoot tips of MM.111, MM.106 and
M.26 root stocks with BA (1.0mg/l). Walkey (1974) observed importance of
polyvinyl pyrolidone (PVP) in liquid culture media to control browning due to
oxidative phenolic compounds and thus to eliminate the inhibitory effect on the
growth of axillary meristem tips. Abbott and Whitely (1976) observed
proliferation of axillary buds from adult apple trees on LS medium (1965).
Seedling cuttings were found to proliferate axillary buds as well as apical bud.
Jones (1976) assessed the effect of phloridizin and phloroglucinol on apple
shoots and reported the origin of new shoots from terminal and lateral
meristems from axillary buds, but not from callus produced at cut ends.
Snir and Erez (1980) cultured shoot segments of apple (cv. M.104,
MM.106 and M.109) possessing lateral buds on Quoirin’s medium (1977) with
vitamins proposed by Wetmore and Sorokin (1955) and observed increase in
axillary shoot proliferation rate. In 1981 Sriskandarajah and Mullins
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successfully produced shoots from axillary buds of Granny Smith cv. in liquid
culture medium. The shoots produced roots in agar free half strength MS liquid
medium. Root formation was promoted by IBA (10µM) and NOA (10µM) but
2,4-D was inhibitory. Constant temperature (26o±2
oC) and continuous light
illumination (10µEm-2S-1) were found essential. Continued efforts in this
direction lead to more success. Broome and Zimmerman (1984) observed
proliferation of lateral buds of apple on MS medium supplemented with BA
(4.4µM), IBA (0.5 – 4.9µM) and GA3 (1.5µM). Better proliferation was
observed on WPM with vigorous growth of shoots but with tissue vitrification.
It was followed by Byeong et al. (1987) who cultured axillary buds of different
cultivars (Empire, McIntosh, Delicious, Triple Red Delicious and Vermount
spur Delicious) on LS (1965) medium supplemented with BA(4.4µM), IBA
(0.5µM) and GA3(1.3µM). All cultivars produced healthy (more than 1 cm
long) shoots in 16 hour photoperiod. At the same time, Yae and Zimmerman
(1987) observed influence of photoperiod, apical meristem and explant
orientation on in vitro axillary shoot prolideration of apple 16 hour photoperiod
with a light intensity of 50-70 uE m-2s-1 was found effective. Laimer (1988)
reported in vitro propagation of aseptically proliferated lateral buds of cv. Graf
Uklhor us Augusthalvill on MS medium. Best growth was observed at BA:
IBA ratio 6:1 and at 24 – 26C with 16 hour light period 8 hour darkness.
Zimmerman and Fordham (1989) observed in apple (cv. Delicious)
culture that more shoots developed from subcultured shoots, if placed inverted
in the medium. Inversion also resulted in decrease in early senescence and
favoured early rooting. Petrovic (1989) in his studies on meristem from five
apple cultivars observed that IBA, BA and GA3 were essential at 0.1, 1 and
0.1mg/l for leaf rosette formation, while lateral bud formation was promoted by
IBA (0.5 mg/l), BA(0.5 mg/l) and GA3(0.1mg/l). In the same year, Ivanova
(1989) who independently reported in vitro morphogenesis of axillary buds of
root stocks - MAK.9, M.26 and MM.106. Good shoot proliferation was
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achieved on a 2-layer nutrient medium (a liquid layer and a modified MS
medium solidified with agar). BA (0.5, 0.7 or 1.0mg/l) promoted axillary
morphogenesis in liquid medium and 66.6 – 100 explants produced lateral
buds. Chen and Evans (1990) obtained multiple shoots from axillary buds of
cv. Golden Crown on MS medium. Correa et al. (1990) cultured sprouts of M.7
root stock, obtained in vitro on MS medium supplemented with IBA (0.0, 1.0,
2.0 and 3.0mg/l) at 25oC under 16 hour light. Best results for rooting were
observed with IBA (1.0 mg/l). Komiya et al. (1991) studied a different
parameter and produced callus from axillary buds under blue light having some
other purpose of obtaining a stain cyanidine-3-galactocide (I) useful as a
colouring agent. On the other hand, Jasik et al. (1997) reported culture of
lateral buds along with cut shoot fragments on media like MS and observed
bud proliferation. Proliferated Shoots resulted in successful in vitro rooting.
III. Stem Section Culture
Stem sections of apple were first cultured in vitro by Saad (1965). He
obtained callus from cut ends of stem sections of apple (Cortland Melntosh) on
White’s medium for use of phytomorphological studies. Later on, Fuji and Nito
(1972) reported callus formation from stem sections of Golden Delicious of
apple. Chong and Taper (1972) also obtained callus from stem sections of some
apple varieties and assessed the role of I. Sorbitol as a carbon, source, in
maintenance of callus growth. In India Mehra and Saroj (1979) recorded that
White's medium supplemented with NAA, kinetin, CW and GA3 was most
effective for callus growth in stem segments of Golden Delicious apple. They
further reported callus differentiation in the form of shoots. However, complete
plantlets were not obtained. Callus formation was also observed by Schneider
(1978) on Fuji and Nito proposed medium (1972) supplemented with myo
inositol (100mg/l) from stem sections of some apple cultivars. Evaldson (1985)
obtained callus from stem segments of variety Akero on MS medium with two
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different concentrations of macronutrients (standard and half strength). The
callus was subjected to differentiation with the help of IBA and BA. BA (5.0
mg/l) stimulated growth and differentiation with low IBA concentration.
Gladyshava (1987) also obtained callus from stem segments of several apple
cultivars and subjected it to differentiation. Complete plantlet formation was
not, however, reported. Kudryarkin and Evseena (1987) cultured small sections
of one year old shoots obtained in October from mature apple trees and got
callus. Callus formation was reduced if the cuttings were exposed to –30C for
12 hours. This observation was followed by Hicks (1987) who reported root
formation from stem cuttings, instead of callus, in root stock selection KSC.3.
Root meristemoids were observed at basal end on MS medium supplemented
with IBA (very low) on 3rd
day. These transformed into root primordia and
finally formed roots.
Morphogenetic response of stem segments of mature apple (cultivars
Akero, McIntosh, McIntosh Wijiik, Gravenstain and root stock M.26) was
observed by Welender (1988). The overall response on N6 modified medium
was poor but only McIntosh Wijiik produced rootless shoots. Belaizi et al.
(1991) initiated a different line of investigation on apple by inducing direct
organogenesis on intermodal segments of in vitro grown shoots of Golden
Delicious apple. They observed best response in terms of number of shoots and
shoot growth potential on MS+BA (4.4µM)+TIBA(1.0µM). It was 1992 when
Pawlicki and Welender observed effect of BA and GA3 on in vitro rooting of
stem discs of apple (M.9 Jork) BA treatment strongly inhibited rooting and
IBA promoted it. Same year Puente and Marin (1992) cultured micro-shoots
after splitting, on suitable nutrient media and observed 71.4% rooting. Best
results were obtained by splitting shoots along their whole length. All split
shoots showed normal growth after rooting and planting in soil. Root
regeneration from basal regions of 95% stem discs of M.4 Jork cv. on nutrient
medium augmented with IBA (24.6µM) was observed by Welender and
Ph.D. Thesis of Wilayat Rizvi E.mail_ [email protected]
C.O.R.D. University of Kashmir, J & K
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50
Pawlicki (1993). Anatomical study of the stem discs revealed the development
of cambium around vascular bundles on 3rd
day of incubation and formation of
root primordia on 7th
day.
Piccionic (1996) developed storage technique for in vitro grown shoots.
Cuttings were excised from micropropagated shoots of apple and were covered
with alginate matrix containing activated charcoal and growth regulators in
addition to essential nutrients. Viability of the shoot cuttings was maintained
without addition of growth regulators even after 4 months and in vitro growth
was resumed on suitable media. A report published by Hyae et al. (1996)
reveals that the workers assessed the impact of, light conditions and pre-
treatment of internodal shoot cuttings on shoot regeneration of Malus
domestica Borkh und