Variability in Response of Potato (Solanum tuberosum) Cultivars to in vitro Shoot Regeneration

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Variability in Response of Potato ( Solanum tuberosum ) Cultivars to in vitro Shoot Regeneration

Mohammed Abdulaziz Al-Sulaiman

Community College at Hreimla, Hreimla 111962, P.O. Box 300, King Saud University, Riyadh, Saudi Arabia

Abstract. This investigation was conducted to establish a reliable in vitro plant regeneration system, which is a prerequisite step before conducting any genetic manipulation system. Five different potato cultivars (Daraga, Sponta, Diamont, Cillan and Burun) with two explants and four medium protocols were used in this study to find out the best cultivar of potato linked with the optimum medium conditions for callus induction; and subsequently plant regeneration. The results indicated that all in vitro traits were highly significant influenced by differences in potato cultivars, explants, and medium protocols, as well as for the interaction between the three different factors, except for the interaction between explants and medium protocols for callus induction and callus weight, which showed no significant difference.

Also, the callus weight was not significantly different due to medium protocols. The shoot formation derived from callus of nodal explants produced the highest percentage of shoot formation (20.7%) across the cultivars, which was significantly different from the shoot formation derived from callus of leaf explants (6.5%). Results also revealed that there was a highly significant interaction between the cultivars and explants, for instance, the Daraga cultivar produced high percentage of shoot formation derived from callus of nodal explants (40.5), while the percentage of shoot formation derived from callus of leaf explants was (7.9%). Results showed that the medium protocol "3" produced the highest percentage of shoot formation (23.9%) across the potato cultivars, which was significantly different from other medium protocols. Results also revealed that there was a highly significant interaction between the cultivar and medium protocol. The Daraga cultivar produced the highest percentage of shoot formation (51.2%) on medium protocol "3", which was significantly different from the other protocols. Results showed that there was a highly significant difference between medium protocols and explants. The highest

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percentage of shoot formation was obtained with callus derived from nodal explants (38%) on medium protocol "3". However, the percentage of shoot formation derived from callus of leaf explant on the same medium protocol was (10%).

1. Introduction

Potato (Solanum tuberosum L.) is a herbaceous annual that grows up to 100 cm tall and produces a tuber - also called potato - so rich in starch, and ranks as the world's fourth most important food crop, after maize, wheat and rice. Potatoes are rich in carbohydrates, making them a good source of energy. They have the highest protein content (around 2.1 percent on a fresh weight basis) in the family of root and tuber crops, and protein of a fairly high quality, with an amino-acid pattern that is well matched to human requirements. They are also very rich in vitamin C - a single medium-sized potato contains about half the recommended daily intake - and contains a fifth of the recommended daily value of potassium.

Micropropagation in potato has been noticed to be a regular essential work followed almost with all transformation, in vitro propagation, microtuberization, and plant regeneration protocols, using basal MS (Murashige and Skoog, 1962) with no or very slight modifications, and suits different potato explants (Romano, et al., 2001;

Banerjee, et al.,2006; Das, 2006; Mullins, et al., 2006; Orczyk, et al., 2007).

Potatoes have been shown to be easily regenerated from leaf tissue (Park, et al., 1995), but their responses to published regeneration regimes have shown cultivar specificity (Wheeler, et al., 1985; Cearley and Bolyard, 1997). Recently, callus induction and plant regeneration in vitro in potato have been studied (Omidi and Shahpiri, 2003). Potato regeneration derived from callus has been successfully employed to serve many potato transformation processes (Beaujean, et al., 1998; Romano, et al., 2001; Davidson, et al., 2004; Craig, et al., 2005; Gustafson, et al., 2006; Orczyk, et al., 2007; Badr, et al., 2008).

The objective of the present study was to establish a reliable in vitro plant regeneration system, which is a prerequisite step before conducting either any potato transformation or in vitro selection.

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2. Materials and Methods

2.1 Plant Material and Source of Explant

Five different potato cultivars (Daraga, Sponta, Diamont, Cillan and Burun) were used in this study. Potato tubers were left in open air until dormancy period was broken, and new sprouts began to bud.

Thereafter, 4-6 weeks old new sprouts budding out from the potato tubers have been used as explant portions to establish the micropropagation technique. The objective of this technique was to maintain the explant material over the year, and subsequently, using the material for potato in vitro culture and plant regeneration from callus.

2.2 Preparation of Potato Culture Media

Composition of the medium protocol used for micropropagation and root induction is shown in Table 1. Full details of the four medium protocols (A, B, C, and D) used for callus induction; differentiation and root induction are given in Table 2.

Table 1. Composition of the medium protocol used for micropropagation and root induction.

Protocol Components Concentration on medium (mg/l)

Inorganic salts MS Salts (1X)

Glycine 2 mg/L

Inositol 100 mg/L

Nicotinic Acid 0.5 mg/L

Pyridoxine HCl 0.5 mg/L

Thiamine HCl 0.1 mg/L

Ascorbic Acid** 40 mg/L

Casein Hydrolysate 500 mg/L

Sucrose 30 gm/L

Agar 6-8 gm/L

For all medium protocols, the pH was adjusted to be between 5.8-6, using 1.0 M HCl or 1.0 M NaOH. Agar was supplied after adjusting the pH by a constant 6-8 gm/L. Sucrose was added by a varied percentage according to the used protocol.

All equipments and media were sterilized for 20 min. at 121°C (15 PSi nominal steam pressure with an extended 30 min. cycle time which was adequate).

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Table 2. Composition of the four medium protocols used for callus initiation and plant regeneration in potato cultivars.

Protocol A (Gynheung et al., 1986)

Protocol Components Protocol sequence / liter

Callus induction Callus differentiation Inorganic salts MS Salts (1X) MS Salts (1X)

Glycine 2 mg/L 2 mg/L

Inositol 100 mg/L 100 mg/L

Nicotinic Acid 0.5 mg/L 0.5 mg/L Pyridoxine HCl 0.5 mg/L 0.5 mg/L

Thiamine HCl 0.1 mg/L 0.1 mg/L

NAA 5 mg/L 0.02 mg/L

BAP 0.1 mg/L

Zeatin Riboside - 2.2 mg/L

GA3 - 0.15 mg/L

Sucrose 16 gm/L 16 gm/L

Agar 6-8 gm/L 6-8 gm/L

Protocol B (Banerjee, et al., 2006)

NAA 0.2 mg/L -

BAP 2 mg/L 2 mg/L

GA3 5 mg/L 5 mg/L

Protocol C (Romano, et al., 2001)

Zeatin (Sigma^®,USA) 0.8 mg/L 0.8 mg/L

2,4-D 2 mg/L -

GA3 - 2 mg/L

Sucrose - -

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Table 2. Contd.

Protocol D (Beaujean, et al., 1998)

Protocol Components Protocol sequence/liter

Callus induction Callus differentiation*

Inorganic salts MS Salts (1X) MS Salts (1X)

Nicotinic Acid 0.5 mg/L 0.5 mg/L

Pyridoxine HCl 0.5 mg/L 0.5 mg/L

NAA 0.5 mg/L -

Kinetin 1 mg/L 1 mg/L

2.3 Culture Preparation and Conditions 2.3.1 Micropropagation

Sprouts were carefully cut under aseptic conditions in a Laminar flow Cabinet; washed with tap water several times (sometimes containing a detergent to decrease the surface tension leading to get more efficient washing), then washed finally with distilled water. Sprouts sterilization was done by dipping them in 0.1% HgCl2 for 15 minutes [sometimes using Cl₂: distilled water (1:4)], then dipping in 70% ethanol for 1 minute, then finally washing 4-6 times with sterilized distilled water.

Sprouts were thereafter cultured aseptically in Standard transparent plastic autoclavable tubes (Falcon Blue Max, 50ml conical, Lincoln Park, New Jersey, U.S.A) containing approximately 10-15ml of culture medium (Table 2), and transferred to the culture room at 25+2 ^ºC on 16 hours illumination (2,000 lux, day light florescent tube lamps).

2.3.2 Callus Induction

Two explants, leaves and node segments, obtained from the micro propagated plantlets, were used for callus induction in all cultivars. The explants were cultured on four medium protocols (Table 2).

Leaves have been aseptically cut into four equal parts (about 2 mm

× 2 mm each), then 10 leaf explants were cultured in standard 10-cm Petri dishes containing approximately 25 ml of culture media (Table 2).

The stem was aseptically cut into fragments to get nodes (about 8 mm length each), then 5 nodal explants were cultured in standard 10 cm Petri

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dishes containing approximately 25 ml of culture media (Table 2). Both cultured leaves and nodes were transferred to the culture room at 25+2

OC on 16 hours illumination (2,000 lux, day light florescent tube lamps).

2.4 Plant Regeneration Derived from Callus

The calli induced from leaf and node explants were then transferred aseptically to be cultured on a shoot inducing media in standard 10-cm Petri dishes containing approximately 25 ml of culture media (Table 2), then transferred to the culture room at 25+2 ^OC on 16 hours illumination (2,000 lux, day light florescent tube lamps).

The differentiated shoots derived from calli, have been aseptically separated from being adhered with the callus parts and transferred to be cultured on the micropropagating medium (Table 1) for root induction in Standard transparent plastic autoclavable tubes (Falcon Blue Max, 50 ml conical, Lincoln Park, New Jersey, U.S.A) containing approximately 10- 15 ml of culture medium to form new roots and multiply, then transferred to the culture room at 25+2 ^OC on 16 hours illumination (2,000 lux, day light florescent tube lamps).

2.5 Callus Induction Response

After 4-5 weeks of incubation, callus induction response was recorded and the following parameters were determined for each Petri dish: a) Callus weight (mg)/explants, b) Callus induction percentage.

2.6 Morphogenetic Response

Calli derived from leaf and node explants were transferred to differentiation media (Table 2) and the percentage of calli with green shoots that exceeded 1 cm in length was recorded.

2.7 Root Induction

The regenerated shoots were transferred to the root induction medium (Table 1).

2.8 Transferring the in vitro Grown Plantlets to Soil (Adaptation Process)

Regenerated plantlets of 5-6 weeks old were washed with tap water to remove agar from roots, and then transplanted in small pots filled with peatmoss: perlite (1:2). As the pots were then transferred to the greenhouse to be incubated and adapted to field conditions.

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2.9 Statistical Analysis

Data were analyzed as a factorial CRD arrangement of cultivars, medium protocols and two different explants (leaves and nodes) in complete randomized design with five replicates. All in vitro traits data, except the callus weight, were subjected to arcsine transformation prior to statistical analysis. Comparison between means was made via the least significant differences multiple range. Data were analyzed using SAS program (SAS Inc., 1985).

3. Results and Discussion

3.1 Micropropagation of Potato Cultivars

Potatoes can be micopropagated rapidly and on a large scale by meristem and shoot tip culture (Goodwin, et al., 1980), proliferated by axillary shoots developed from in vitro cultured nodal cuttings (Hussey and Stacey,1981), and in vitro mass tuberization (Wang and Hu, 1985).

In the present investigation, the micropropagation technique facilitates the production and conservation of potato cultivars in controlled disease free conditions and to obtain many shoots over the year for callus induction and plant regeneration system.

The shoot multiplication of the five potato cultivars were propagated in vitro by placing nodes from sprouted tubers on Murashige and Skoog medium without hormone (Fig. 1A). In vitro plantlets spontaneously formed roots on agar culture medium (Fig. 1A). Plantlets lifted in culture tubes for two months without subculture, formed small microtubers (Fig. 1B).

Plantlets were transferred to pots containing peatmoss: perlite (1:2) for adaptation and subsequently whole plant system was obtained under green house conditions (Fig. 1C, 1D).

One way of conserving germplasm, an alternative to seed banks and especially to field collections of clonally propagated crops, is in vitro storage under slow-growth conditions (at low temperature and/or with growth-retarding compounds in the medium) or cryopreservation or as desiccated synthetic seed ( Villalobos and Engelmann, 1995).

3.2 Response of Explant to Callus Induction and Plant Regeneration Establishing reliable in vitro plant regeneration is a prerequisite step before conducting either any potato transformation experiment or in

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vitro selection. If particular plant species shows no competence for plant regeneration, the chances will be regeneration from useful cell lines in the same species may also be unsuccessful. Once a tissue culture system is capable of plant recovery has been established, the next step is to apply the developed system to potato genetic manipulation studies. Having this as a principle, the present work was initiated with the aim of finding a tissue culture system competent to regenerate plants from cultured tissues of potato.

Fig. 1. In vitro propagation of potato. (A) Shoot multiplication and root induction. (B) Microtubers produced in vitro. (C) Plant regeneration for adaptation. (D) Whole plant under green house conditions.

This section presents the results of experiments designed to find out the best cultivar of potato linked with the optimum medium conditions for callus induction; and subsequently plant regeneration. Results of these experiments could be presented as follows:

3.3 Callus Induction

Data of callus production, derived from leaf and nodal explants sourced from the five potato cultivars, were recorded after 4-6 weeks

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incubation. These explants were incubated on media previously developed and successfully employed by other investigators for potato.

Analysis of Variance (ANOVA) presented in Table 3 indicated that callus induction was highly significantly influenced by differences in potato cultivars, explants, and medium protocols, as well as for the interaction between the three different factors, except for the interaction between explants and medium protocols, which showed no significant difference.

Table 3. Mean squares from the analysis of variance for in vitro culture traits in potato.

Source of

Variance D.F.

M.S. for in vitro culture traits Callus

induction (%)^a

Callus weight (mg)

Shoot formation (%)^a

Root formation (%)^a Cultivar (A) 4 4518.7** 151626.5** 1394.0** 674.8**

Explant (B) 1 6099.6** 541981.0** 8954.7** 4734.4**

Protocol (C) 3 718.0** 18093.6^NS 2015.0** 1395.8**

A × B 4 2223.3** 148916.6** 486.5** 643.0**

A × C 12 1172.8** 34250.0** 383.0** 428.3**

B × C 3 73.0^NS 25546.7^NS 542.6** 1165.2**

A × B × C 12 376.0* 34215.0** 274.6* 414.6**

Error 160 199.7 12364.2 150.6 81.1

a: Data were subjected to square root transformation.

**: Highly significant at 0.01 probability level.

*: Significant at 0.05 probability level.

NS: Not Significant at 0.05 probability level.

Callus formation was clearly varied among the five cultivars regarding the two explants as shown in Table 4a, by which the percentage of the leaf explants that succeeded to develop calli has been ranged from 32% (Burun) to 68% (Sponta). On the other hand, the percentage of nodal explants that succeeded to develop calli ranged from 39% (Cillan) to 89.5% (Daraga).

Growth rate of callus was dependent upon cultivar and explants.

The cultivar Spunta produced the highest percentage of callus induction (69.2%) across the explants, which were not significantly different from the cultivar Daraga (67.3%) (Table 4a).

On the other hand, the nodal explant produced the highest percentage of callus induction (61.5%) which was significantly different from the percentage of callus induction, derived from leaf explants (46.8%). It is evident from Table 3 that the interaction between cultivars and explant was highly significant. The cultivar Daraga gave high

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percentage of callus induction with nodal explants, which was significantly different from the percentage of callus induction derived from leaf explants (Table 4a).

The response of callus induction was affected by the used medium.

Table 4(b) indicates that the medium protocol "2" gives the highest average of callus induction reaching 57.7% across genotypes. However, it was not significantly different from the medium protocol "1"and the medium protocol "3". While the lowest average of callus induction was given by the medium protocol "4" of 47.2%. The interaction was highly significant between cultivars and medium protocols. The cultivar Spunta gave the highest callus induction (92.1%) with medium protocol "2".

However it had the significantly lowest callus induction percentage (47.3%) when its explants were cultured on medium protocol "4" (Table 4b).

The color of callus was dependent upon the cultivar and the culture medium employed. The callus was yellow to yellowish green and began to develop green regions after two weeks of incubation (Fig. 2).

Table 4(a). Means of callus induction (%) as influenced by cultivars and explants and their interaction.

Cultivars Explant

Cultivar Means Leaf Node

Daraga 45.2 89.5 67.3 A

Sponta 68.0 70.6 69.2 A

Diamont 48.8 59.2 54.0 B

Cillan 40.0 39.0 39.4 C

Burun 32.0 49.5 40.7 C

Explant Means 46.8 B 61.5 A L.S.D. 0.05 for cultivar means = 8.67

L.S.D. 0.05 for explant means = 5.49

L.S.D. 0.05 for cultivar x explant means = 12.29

Table 4(b). Means of callus induction (%) as influenced by cultivars and medium protocols and their interaction.

Cultivars Medium Protocols

A B C D

Daraga 73.0 63.4 57.0 76.0

Sponta 65.8 92.1 71.7 47.3

Diamont 66.0 69.0 46.5 34.6

Cillan 34.6 36.6 36.6 50.0

Burun 48.3 27.6 59.1 28.0

Protocol Means 57.5 A 57.7 A 54.2 AB 47.2 B L.S.D. 0.05 for cultivar x protocol means = 17.39

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3.4 Callus Weight

Callus growth and development were influenced by a complex relationship between explant used to initiate the callus, the constitutes of medium protocols, and cultivars. Analysis of variance (Table 3) indicates that callus weight was highly significant influenced by differences in potato cultivars and explants and their interactions as well as the interaction between the cultivars and medium protocols (Table 3).

Fig. 2. Plant regeneration derived from callus of potato :(A)Callus derived from nodal explants, (B): Callus derived from leaf explants, (C) Shoots and roots derived from callus.

Growth rate of callus was dependent upon the cultivar. The cultivar Daraga produced the highest callus weight (208.3 mg/explant) across explants used (Table 5a), and was significantly different from all other cultivars. On the other hand, the cultivars Cillan had the significantly lowest callus weight (41.1 mg/ explant). It is evident from Tables 3 and 4(a) that the interaction was highly significant between cultivars and explants. The cultivar Daraga gave the highest response to callus weight (369.0 mg/ explant) with nodal explants used, which was significantly different from the callus weight derived from leaf explants (Table 5a).

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The interaction was highly significant between cultivars and medium protocols (Tables 3 and 5 b). The cultivar Darga gave the highest callus weight (336.1 mg/ explant) with medium protocol "2". However, it had the significantly lowest callus weight (76.6 mg/ explant) with medium protocol "4" (Table 5b).

In conclusion, it can be suggested that before utilizing tissue culture techniques as a tool for crop improvement, it is necessary to determine the factors influencing callus formation, its quality and quantity during induction and maintenance and subsequently shoot regeneration from callus. The previous results provided an indication about the relative importance of genotypes explants and media protocols in culture response. These results showed that the callus initiation and its growth rate were dependent upon the genotype and culture medium employed. Differences have been recorded among potato cultivars (Ovesna, et al., 1993; Dale and Hampson, 1995; Jaysree, et al., 2001).

3.5 Shoot Formation

Statistical analysis of shoot formation derived from callus revealed highly significant difference among potato cultivars, explants, and medium protocols (Table 3). All the two way interactions were highly significant. However, the second order interaction (cultivar × explant × protocol) was significant (Table 3).

Table 5(a). Means of callus weight (mg) as influenced by cultivars and explants and their interaction.

Cultivars Explant

Cultivar Means

Laef Node

Daraga 47.6 369.0 208.3 A

Sponta 54.7 113.7 84.2 BC

Diamont 93.8 152.1 123.0 B

Cillan 14.97 67.3 41.1 C

Burun 98.1 127.6 112.8 B

Explant Means 61.8 B 166.0 A

Table5(b). Means of callus weight (mg) as influenced by cultivars and medium protocols and their interaction.

Cultivar Medium Protocols

A B C D

Daraga 190.7 336.1 229.6 76.6

Sponta 75.8 53.2 102.6 105.3

Diamont 79.0 135.0 112.7 165.3

Cillan 39.4 72.0 26.2 27.0

Burun 119.0 114.6 59.7 158.2

Protocol Means 100.8 A 142.1 A 106.1 A 106.5 A L.S.D. 0.05 for cultivar x protocol means = 98

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Results in Table 6(a) showed that the cultivar Daraga produced the highest mean value of shoot formation (24.2%) across explants, which was significantly different from the other cultivars. The shoot formation derived from callus of nodal explants produced the highest percentage of shoot formation (20.7%) across the cultivars, which was significantly different from the shoot formation derived from callus of leaf explants (6.5%) (Table 6a). Results also revealed that there was a highly significant interaction between the cultivars and explants for instance, the cultivar Daraga produced high percentage of shoot formation derived from callus of nodal explants (40.5%), while the percentage of shoot formation derived from callus of leaf explants was (7.9%) (Table 6 a).

Results in Table 6(b) show that the medium protocol "3" produced the highest percentage of shoot formation (23.9%) across the potato cultivars, which was significantly different from the other medium protocols. Results also revealed that there was a highly significant interaction between the cultivars and medium protocol. The Daraga cultivar produced the highest percentage of shoot formation (51.2%) on medium protocol "3", which was significantly different from the other protocols.

Results of Table 6(c) show that there was a highly significant difference between medium protocols and explants. The highest percentage of shoot formation was obtained with the callus derived from nodal explants (38%) on medium protocol "3". However, the percentage of shoot formation derived from callus of leaf explant on the same medium protocol was (10%).

In conclusion, the ability of fresh callus to differentiate into plantlet depends on the hormone level of the initial callus induction medium, as well as on the genotype of the donor plant. Factors considered to be important for eliciting success in morphogenesis have been listed by Thorpe (1980). These include: (a) Selection of the organ to be used as a tissue culture source, (b) The appropriate physiological and ontogenetic age of the organ, (c) The suitable season in which the explant is obtained, (d) The size of explant, and (e) The overall quality of the plant from which explants are derived.

Results from the present investigation showed that there was significant genotype×medium interaction for morphogenetic response.

The probable reason of differences in morphogenetic response in vitro

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may be attributed to (a) Genetic differences among the used genotypes, (b) Differences in the growth regulators, or (c) Differences in the growth conditions and age of the source of explants.

Table 6(a). Means of shoot formation (%) as influenced by cultivars and explants and their interaction.

Cultivar Explant

Cultivar Means L N

Daraga 7.9 40.5 24.2 A

Sponta 13.3 19.6 16.5 B

Diamont 5.1 19.0 12.1 BC

Cillan 2.3 8.6 5.5 D

Burun 3.8 15.8 9.8 CD

Explant Means 6.5 B 20.7 A

Table 6(b). Means of shoot formation (%) as influenced by cultivars and medium protocols and their interaction.

Cultivars Medium protocols

A B C D

Daraga 18.0 9.6 51.2 18.0

Sponta 9.3 24.1 25.7 6.7

Diamont 10.4 12.3 13.6 12.0

Cillan 5.3 2.0 10.6 4.0

Burun 5.3 10.0 18.6 5.3

Protocol Means 9.7 B 11.6 B 23.9 A 9.2 B Table 6(c). Means of shoot formation (%) as influenced by medium protocols and explants

and their interaction.

Protocol Explant

L N

Protocol 1 4.0 15.3

Protocol 2 6.8 16.4

Protocol 3 10.0 38.0

Protocol 4 5.2 13.2

L.S.D. 0.05 for cultivar x explant means = 7.63 L.S.D.0.05 for cultivar x protocol means = 10.8 L.S.D.0.05 for explant x protocol means = 6.8

The potential of an isolated undifferentiated plant cell to regenerate into a plant of potato explants has been investigated by many researchers (Gahan, 2007; Gustafson, et al., 2006; & Orczyk et al., 2007). They reported that the process of callus induction and plant regeneration in potato could be achieved by applying different protocols with a variety of employed explants (leaf, nodes, internodes, and tuber discs) among different genotypes; revealing that potato is a very flexible plant during handling in vitro. Also, many differences have been recorded among

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potato cultivars in their ability to regenerate adventitious shoots (Ovesna, et al., 1993 & Dale and Hampson, 1995).

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