review of literature - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/3362/7/07_chapter...

Chapter – II

Review of Literature

Ph.D. Thesis of Wilayat Rizvi E.mail_ [email protected]

C.O.R.D. University of Kashmir, J & K

9

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



10

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



11

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



12

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



13

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



14

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



15

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



16

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



17

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



18

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)



19

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



20

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



21

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



22

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



23

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



24

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



25

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



26

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



27

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



28

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



29

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



30

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



31

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



32

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,



33

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



34

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



35

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



36

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



37

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.



38

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



39

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



40

(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



41

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



42

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



43

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



44

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).



45

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.



46

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



47

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



48

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



49

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



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

review of literature - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/3362/7/07_chapter...

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