CA2469139A1 - Potato regeneration system and use thereof - Google Patents

Abstract

The present invention is a medium and a protocol for regenerating potato plants from axenic explants. The method is a one step method and can be applied to a range of explants including shoots, leaves and petioles. The protocol can be used in conjunction with Agrobacterium-mediated transformation to provide a high efficiency transformation method.

Description

Potato Regeneration System and Use Thereof Field of the Invention:
This invention relates to the regeneration of plants from selected tissue from a donor plant. More specifically, it is a one step shoot regeneration system for potatoes.
Background of the Invention:
Regeneration systems are used routinely in the production of seed potatoes and the production of transgenic potatoes. With regard to transgenic plant production, the most limiting factor in their production is the establishment of an efficient protocol for plant regeneration from transgenic tissue. Shoot regeneration in potato (Solanum tuberosum L.) depends primarily on the genotype, medium composition, explant tissue source and cultivation condition.
Examples of media compositions can be found in DeBlock, 1988; Sheerman and Bevan, 1988;
Stiekema et al., 1988; Wenzler et al., 1989. While one with no knowledge of the art would be unaware that subtle changes to a medium result in unpredictable outcomes, one skilled in the art would be very aware of this fact. Hence, they would recognize that changing a known medium even if slightly 2o could result in a non-obvious and unpredictable result.
In general, stem sections are used as the explant source. As regeneration efficiency is partially dependent upon explant source, one generally is required to develop specific media for specific explant tissue sources. This therefore restricts the amount of starting tissue that can be obtained from a plant for use in a given protocol. While this may be of little significance for seed potato production of standard cultivars, it could be very significant for scale up of transgenic and rare plant materials.
In general, there is a strong interaction between the various factors controlling 3o regeneration efficiency. This therefore requires that specific media be developed for each set of factors. As there are at least three factors that interact with the medium to determine regeneration efficiency, it is clear that there is a need for many different media for each step in the regeneration process.
Most procedures for regeneration are mufti-step, wherein there are specific media for, as an example, callus formation and shoot formation. This requires that the tissue be handled a number of times, thereby increasing the chance of contamination. More significantly, the cost of multiple steps is high - each medium must be prepared from stock solutions that frequently contain costly growth hormones and of course, the preparation of medium, and transfer of tissue increases the manpower expenses tremendously. Hence, it is an objective of the invention to overcome the deficiencies in the prior art.
Summary of the Invention:
The present invention provides an efficient multipurpose, regeneration medium and protocol for using the medium to regenerate potato plants from explants. The medium and protocol have advantages over others including the fact that they can take advantage of the morphogenetic capacity of potato petioles, there is significantly increased regeneration efficiency, there is reduced time between placing explants onto regeneration medium and shoot formation, there is a low level of somaclonal variations, and there is a high recovery rate after 2o transferring shoots to a rooting medium. In addition, it is a one-step regeneration protocol in contrast to classic DeBlock's [ 11 ] protocol for potato regeneration where different media are used at certain stages of potato cultivation. Further the protocol is relatively genotype independent.
In one embodiment of the invention, a regeneration medium for potato is provided comprising:
MS salts;
one of MS vitamins or BS vitamins;
a concentration of 30-50 mg/1 of adenine' 504;
a concentration of 18-22 g/1 of glucose;
a concentration of 18-22 g/1 of mannitol;
3o a concentration of 700-1500 mg/1 of MES;
a concentration of 0.03-0.05 mg/1 of gibberelic acid (GA3);

a concentration of 0.015-0.03 mg/1 of NAA;
and a concentration of 1.5-5 mg/1 of zeatin riboside;
and the medium is adjusted to a pH of 5.6-5.8 with 1N KOH.
In one aspect of the invention, the medium further comprises solidified agarose, and the agarose concentration ranges from 3.3 to 4.2 g/1.
In another aspect of the medium of the invention, the agarose concentration is about 4 g/l.
1o In another aspect of the medium of the invention, the glucose concentration is about 20 g/l.
In another aspect of the medium of the invention, the mannitol concentration is about 20 g/1.
In another aspect of the medium of the invention, the GA3 concentration is about 0.04 mg/l.
In another aspect of the medium of the invention, the NAA concentration is about 0.02 mg/l.
In another aspect of the medium of the invention, the zeatin riboside concentration is about 2 mg/l.
In another embodiment of the invention a combination for regenerating potato plants is provided.
The combination comprises a medium, and an axenic explant. The medium comprises:
MS salts;
one of MS vitamins or BS vitamins;
a concentration of about 30-50 mg/1 of adenine' S04;
a concentration of about 18-22 g/1 of glucose;
a concentration of about 18-22 g/1 of mannitol;
a concentration of about 700-1500 mg/1 of MES;
a concentration of about 0.03-0.05 mg/1 of gibberelic acid (GA3);
3o a concentration of about 0.015-0.03 mg/1 of NAA;
a concentration of about 1.5-5 mg/1 of zeatin riboside;

a concentration of about 3.8-4.5 g/1 agarose;
the medium adjusted to a pH of about 5.6-5.8 with 1N KOH; and the explant comprises one of shoots, petioles and leaves.
In one aspect of the combination of the invention, the explant is a petiole.
In another embodiment of the invention, a one-step regeneration protocol for potato plants is provided. The protocol comprises placing an axenic explant in an axenic medium and culturing the axenic explant under axenic culture conditions. The axenic explant comprises one of leaves, l0 stems and petioles. The axenic medium comprises:
MS salts;
one of MS vitamins or BS vitamins;
a concentration of about 40 mg/1 of adenine' 504;
a concentration of about 20 g/1 of glucose;
a concentration of about 20 g/1 of mannitol;
a concentration of about 900 mg/1 of MES;
a concentration of about 0.04 mg/1 of gibberelic acid (GA3);
a concentration of about 0.02 mg/1 of NAA;
a concentration of about 2 mg/1 of zeatin riboside;
a concentration of about 4 g/1 agarose;
the medium adjusted to a pH of about 5.7 with 1N KOH.
The axenic culture conditions comprising a temperature ranging from 22°
to 26°C with photoperiod ranging from 14 to 18 h light and a light intensity ranging from 2500 to 3200 lux.
In one aspect of the regeneration protocol of the invention, the axenic explant is a petiole.
In another embodiment of the invention, a use of a regeneration medium for the one step regeneration of potato plants from an axenic explant is provided. The regeneration medium comprises:
3o MS salts;

one of MS vitamins or BS vitamins;
a concentration of about 40 mg/1 of adenine' 504;
a concentration of about 20 g/1 of glucose;
a concentration of about 20 g/1 of mannitol;
a concentration of about 900 mg/1 of MES;
a concentration of about 0.04 mg/1 of gibberelic acid (GA3);
a concentration of about 0.02 mg/1 of NAA;
a concentration of about 2 mg/1 of zeatin riboside; and a concentration of about 4 g/1 agarose;
1o and the medium is adjusted to a pH of about 5.7 with 1N KOH.
In one aspect of the invention, the use further comprises transforming said axenic plant by Agrobacterium-mediated transformation.
Figure Descriptions:
Figure 1. Multiple shoot regeneration from potato petioles after 30 (A) and 40 days (B) of cultivation on PetM medium. Bar=t cm.
Detailed Description of the Invention:
Example 1:
Developing regeneration protocols:
We developed and tested many different media in order to establish a high frequency regeneration protocol for Solanum tuberosum L. As a source of plant tissue we used 4-5-week-old axenically grown potato plants cvs Desiree Zarevo, Lvivyanka, Lugivskiy, and Svitanok kyivskiy. The range of media components included MS salts (Murashige and Skoog, 1962), MS
or Gamborg's vitamins (Gamborg et al., 1968), 0-100 rng L-1 of adenine . 504, 5-30 g L-1 of 3o glucose, 5-30 g L-1 of sucrose, 0-60 g L-1 of mannitol, 0-2000 mg L-1 of 2-[N-MorpholinoJethanesulfonic acid (MES), 0-5 mg L-1 of GA3, 0-5 mg L-1 of 3-indoleacetic acid (IAA), 0-5 mg L-1 of naphthaleneacetic acid (NAA), 0-10 mg L-1 of 6-benzylaminopurine (BA), 0-10 mg L-1 of zeatin riboside (ZR), and 3-7 g L-1 of agarose at pH5.6-5.8.
Growth regulators GA3 and ZR were filter sterilized and added to the autoclaved medium cooled to 400C. The following explants were tested: stem segments; petioles; and leaves. The whole plants were placed into liquid regeneration medium, cut into 5-15 mm full-length pieces of internodular stem segments or petioles, and then cultivated horizontally onto the same, but solid, medium. Leaves (8-12 mm) were cut at the base and placed on the medium upside up. The upper surface of some leaves was gently wounded with sandpaper to stimulate callus and regeneration response.
Cutting the plants submerged in liquid medium prevented excessive drying of the excised tissue 1o and increased the viability of the explants. Callus formation and subsequent shoot regeneration occurred mainly on the cut/wounded parts of plant tissue. This is important for successful Agrobacterium-mediated transformation.
Among the carbohydrates tested, glucose at concentration of 20 gl 1 was the best carbohydrate source for inducing th~ formation of compact green callus. This type of callus is most favorable for the induction of shoot organogenesis in potato. In the presence of sucrose, the explants formed green and white callus, often friable, with significantly lower regeneration rate comparing to the medium with glucose. Adding mannitol (20 gl 1 ) to the regeneration medium with glucose had positive effect on forming dense, favorable for shoot regeneration cell colonies.
2o Among the cytokinins tested, the best regeneration response from all types of explants was obtained on the medium with zeatin riboside. Interestingly, leaf explants required higher concentration of zeatin riboside to achieve maximum rate of plantlet production (5 mgl-1 vs 2 mgl 1 for stem segments and petioles), although some regenerated shoots looked vitrificated.
This could be related to different levels of exogenous hormones in the explants. The medium with zeatin riboside provided 100% regeneration efficiency with multiple shoots per each explant.
When benzyl adenine phosphate (BAP) was used, all explants produced callus and regenerated shoots as well, but induction of shoot development occurred I-2 weeks later 3o comparing to the medium with the same concentration of zeatin riboside;.
Also the average number of shoots per explant was a bit lower. Further, the shoots regenerated on the medium with BAP were smaller in diameter, and their viability and survival rate after transferring to the rooting medium was lower in contrast to those ones regenerated on the medium with zeatin riboside.
Addition of gibberrelic acid to regeneration medium (0.04 mgl 1 ) stimulated shoot elongation both in the medium with BAP and zeatin riboside.
Among the auxins tested, napthalene acetic acid (NAA) was superior to indole acetic acid (IA.A) with optimum concentration of 0.02 mgl 1. This is several times lower than normally used in other protocols on potato regeneration.
Summarizing the data from different media compositions, the best shoot regeneration response from all types of explants was obtained on the Medium C (Fig. l ). ).
Other regeneration 1s media that produced some regeneration included the following:
MS salts, Gamborgs vitamins, 30 g/1 sucrose, 900 mg/1 MES, 0.1 mg/1 NAA, 5 mg/1 ZR, pH5.7;
MS salts, Gamborgs vitamins, 20 g/1 glucose, 20 g/1 mannitol, 900 mg/1 MES, 0.04 mg/1 GA3, 0.1 mg/1 NAA, S mg/1 ZR, pH5.7; and 2o MS salts, Gamborgs vitamins, 20 g/1 glucose, 20 g/1 mannitol, 900 mg/1 MES, 0.04 mg/1 GA3, 0.02mg/1 NAA, 2 mg/1 BA, pH5.7.
The content of this medium is presented in below in "Experimental". The first regenerated shoots were identified after 12 days of cultivation. All explants on this medium produced multiple shoots (100% regeneration efficiency) within 2-3 weeks.
Petioles were found 25 to have the best regenerative capacity, following by stem segments and leaves, respectively.
Example 2:
Plant transformation, selection and regeneration Plasmid construction The following three binary vectors were used for potato transformation: pBI121 (Jefferson et al., 1987), pWIN3.12-GUS and pPIN-GUS. All of them are based on plasmid pBI121 and designed to express Li-D-glucuronidase (GUS) gene from different promoters. Bp I121 contains the strong constitutive 35S promoter of the cauliflower mosaic virus (CaMV).
pWIN3.12-GUS contains the 823 by fragment of wound-inducible promoter WIN3.12 from a hybrid poplar tree (Rollick and Gordon, 1993). To make this construct, the promoter part of a proteinase inhibitor-like gene WIN3.12 (GenBank accession # L11233) was amplified in a PCR
reaction from a plasmid pWIN3.12. The plasmid pWIN3.12, which has 1.5 kb HindIII genomic io poplar clone of the WIN3.12 gene in the pBluescript BS+ vector (Stratagene, USA), was kindly given by Prof. M.P.Gordon (University of Washington, Seattle, USA). PCR
amplification was carried out in 100 ~1 reaction mix containing 70 ng of pWIN3.12, 2 Units of Deep Vent DNA
polymerise (New England Biolabs) and standard concentrations of primers, MgCl2 and dNTPs.
Deep Vent DNA polymerise was chosen to minimize incorrect incorporation of nucleotides.
The sequences of the primers used for WIN3.12 promoter amplification were:
forward primer WINS (5'-AACTGCAGAAGCTTCCAACATCAATGAT-3', 28-mer), reverse primer WIN3 (5'-CGGGATCCTCTAGAATTTGTTGAATATGAG-3', 30-mer). The underlined parts of the primers correspond to the WIN3.12 promoter sequence in GenBank Database, HindIII (forward primer) and engineered XbaI (reverse primer) sites are shown in bold and were used in 2o subsequent cloning.. PCR amplification of WIN3.12 promoter was performed with manual hot-start and denaturing the template DNA at 94°C for 3 min, following by 30 repeat cycles of 30s at 94°C, 30s at 55°C, and 1 min at 72°C, with a 10 min extension at 72°C for the last cycle prior to halting the reaction at 4°C. After electrophoresis in a 1% agarose gel, a 846 by PCR product corresponding to the desired length of the WIN3.12 promoter was excised, purified with NucleoSpin Extraction Kit (Clontech, USA), digested with HindIII and XbaI
enzymes, and the 823 by HindIII/XbaI DNA fragment was ligated into the corresponding sites of pBI121 (Clontech, USA) in place of preliminary deleted CaMV 35S promoter. The result was a transcriptional fusion between the WIN3.12 promoter, the GUS coding region and the nopaline synthase polyadenylation signal. The correct insertion and full nucleotide sequence of the 3o amplified WIN3.12 promoter was confirmed by mapping with SacI restriction enzyme and by s DNA sequence analysis.
pPIN-GUS contains ~1 kb promoter sequence of wound-inducible potato proteinase inhibitor II
gene (Thornburg et al., 1987). This construct was made by replacing the CaMV
35S promoter in pBI121 with the HindIII/XbaI fragment from plasmid pRT210 (kindly provided by Prof. R.
Thornburg, Iowa State University, Ames, YSA) containing 5'flanking sequence of the wound-inducible potato proteinase inhibitor IIK gene.
All plasmid constructs were maintained in DHSa E.coli strain and introduced into PM90 Agrobacterium tuinefaciens strain via direct transformation. The presence of the engineered vectors in the antibiotic-selected A.tumefaciens clones was confirmed by PCR
analysis using 1 o prornotor-specific primers.
Bacterial culture A.tumefaciens cultures were grown at 26-28°C on a shaker at 220 rpm in liquid Luria-Bertani (LB) medium (Sambrook et al., 1989) containing 100 mg/1 kanamycin (SigmaTM, USA), 100 mg/1 rifampicin (Sigma, USA) and 10 mg/1 gentamicin (Sigma, USA) to mid-log phase (OD600~.5). The bacterial cells were collected by centifugation at 1500 rpm for 10 min and resuspended in liquid plant medium C to a final cell density of 1.0 (OD600) immediately before cocultivation.
The following explants from 4- to 5-week-old potato plants were used for plant transformation:
internodal stem segments, petioles and leaves. The whole plants were placed in the liquid regeneration medium C, cut with sterile blade into 5-15 mm full-length internodal stem segments or petioles, and precultured by floating on 15 ml of liquid medium C in a 9 cm Petri dish. The leaves (8-12 mm) were cut at the base as described by DeBlock (1988) and placed on the medium upside up. Some of the leaves were gently wounded by sandpaper. After 2 days of pre-cultivation at 24°C under low light intensity (500 lux), the potato explants were immersed in Agrobacterium suspension and incubated for 30 min with slow shaking, then blotted with sterile filter paper to remove the excess bacteria, placed horizontally on antibiotic-free medium C
3o solidified with 0.4%(w/v) agarose and cocultivated at 24°C under low light for 3-4 days. After this period, the infected explants were washed for 30 min with sterile medium C containing 1 g/1 cefotaxime, blotted dry and transferred to selective regeneration medium C
containing 50-100 mg/1 kanamycin and either S00 mg/1 carbenicillin or 500 mg/1 cefotaxime. The explants were cultivated at 24°C with 16 h light photoperiod (3000 lux light intensity) and transferred to fresh antibiotic-containing medium C every 2 weeks. Regenerated shoots (1-1.5 cm high) were excised and rooted in the hormone-free medium used for potato micropropagation (see Plant material) and supplemented with 25 mg/1 kanamycin. Transformation frequency was determined as the percentage of inoculated explants that produced kanamycin-resistant plantlets.
to Polymerase chain reaction (PCR) analysis of transgenic plants Potato genomic DNA was isolated from 100 mg of leaf tissue of kanamycin-resistant transfonmants and a control plant using Sigma GenEluteTM Plant Genomic DNA Kit (Sigma, USA). 200-300 ng of plant DNA (5 ~1 out of 100 ~1 of extracted DNA) were analysed in 50 ~1 of PCR mix containing Taq PCR Master Mix (Qiagen, USA) and specific primers.
PCR
amplifications were carned out with the following parameters: 94°C for 3 min, then thirty cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 1 min 30 s, followed by a final 10 min incubation at 72°C. Specific primers for the plants containing pBI121, pWIN3.12-GUS and pPIN-GUS constructs had 5'- and 3'-end nucleotide sequence of the GUS gene and were designed to amplify 1812 by full-length GUS: forward primer 5'GUS (5'-ATG TTA
CGT CCT
GTA GAA ACC-3', 21-mer) and reverse primer 3'GUS (5'- TCA TTG TTT GCC TCC CTG
CTG - 3', 21-mer). Another set of specific primers for the plants with pWIN3.12-GUS
construct was used to confirm correct promoter+gene fusion (984 by amplified DNA fragment):
forward primer 5'WIN (see "Plasmid construction") and reverse primer 3'M/GUS
with sequence complimentary to nucleotides +89 to +109 of the GUS gene upstream region (5'- CTT
TCC CAC CAA CGC TGA TCA - 3', 21-mer). Amplified products were separated in a 1%
(w/v) agarose gels and visualized by staining with ethidium bromide.
Southern analysis Genomic DNA was isolated as described above. Five wg of potato DNA from each clone were digested with 200 units of XbaI restriction enzyme in 200 p,l of reaction mix.
Complete digestion of DNA samples was verified by running mini-gel with small aliquots (10 pl) of the reaction mix. Before loading the gel for Southern, the DNA was concentrated using SpeedVacTM
Concentrator (Savant USA) to reduce volume of incubation mix to well's size.
The DNA
samples were electrophoresed in a 1% agarose gel using lxTBE buffer (Sambrook et al., 1989), then transferred to a Biodyne B nylon membrane (PALL) by capillary blotting following the manufacturer's instructions for the improved alkaline DNA transfer. Pre-hybridization (3 h) and hybridization (overnight) were carried out at 65°C in 10 ml of PerfectHyb PIusTM hybridization to buffer (Sigma, USA) supplemented by 100 pg/ml of sheared, denatured salmon testis DNA
(Sigma, USA). A 1812 by full-length GUS nucleotide sequence was amplified by PCR from pBI121 plasmid, fractionated by electrophoresis, excised from the gel and purified with NucleoSpin Extraction KitTM (Clontech, USA) for use as a hybridization probe.
The probe was labeled with [a-32P]dCTP to a specific activity of >109 cpm/pg using the random priming 1 s method, and used at concentration of 4x 106 cpm of purified probe in 1 ml of hybridization solution. After hybridization, the membrane was washed with constant agitation two times in 2xSSC, 0.1% SDS at room temperature for 5 min, two times in lxSSC, 0.1% SDS at 50°C for 10 min, and then exposed to X-ray film (Kodak BioMaxTM) overnight at -80°C
using intensifying screen. Also, the membrane was exposed to a Phosphor Screen (Molecular Dynamics, USA) and 20 image was scanned using PhosphorImagerTM and Image QuantTM software (Molecular Dynamics, USA).
Transgenic plant wounding and tissue sampling 25 Leaf tissue samples for protein extraction were collected from the upper part of fully developed young potato leaves on one side of the midrib. After the first samples had been removed, the lower part and the periphery of the sampled leaf (~50% of the leaf) were wounded with lab forceps. After 18 hours, the upper part of the same leaf was sampled as before on the opposite side of the midrib.

GUS assay Potato leaf samples harvested as described above, both prior to and 18 hours after mechanical wounding, were homogenized in lysis buffer containing SO mM sodium phosphate pH7.0, 10 mM EDTA, 0.1 % Triton X-100, 0.1 % sarcosyl and 10 mM 2-mercaptoethanol.
Protein content was measured with a standardized Bradford assay (Bradford, 1976). Quantitative fluorometric assay of GUS activity was performed as described earlier (Jefferson et al., 1987) by incubation of the extracts (20 pg protein) with 1mM 4-methyl-umbelliferyl-~i-D-glucuronide (MUG) in lysis buffer for 60 min at 37°C. GUS activity was calculated as pmol of 4-methyl-umbelliferone (4-1 o MU) produced per minute per mg of soluble protein.
Experimental:
Plant material Solarium tuberosum L. cv Desiree plants were grown axenically in culture tubes (Sigma, USA) on the medium containing MS salt mixture (Murashige and Skoog, 1962), Gamborg's vitamins (Gamborg et al., 1968), 20g/1 of sucrose and 7 g/1 of DifcoTM agar with a final pH5.8, under a 16 h light (3000 lux)/8 h dark photoperiod at 240C.
Medium for potato regeneration (Medium C) One-step shoot regeneration medium C was used for shoot induction and plant regeneration from shoot, petiole and leaf explants of potato. It contained MS salts (Murashige &
Skoog, 1962), MS
vitamins or BS vitamins (Gamborg et al., 1968), 40 mg/1 of adenine ' S04~ 20 g/1 of glucose, 20 g/1 of mannitol, 900 mg/1 of MES, 0.04 mg/1 of gibberelic acid (GA3), 0.02 mg/1 of NAA, 2 mg/1 of zeatin riboside, 4 g/1 of agarose. The pH was adjusted to 5.7 with 1N KOH.
Growth regulators GA3 and zeatin riboside were filter sterilized and added to the autoclaved medium cooled to 40-SOOC.

Culture conditions The explants were cultured at 24°C with 16 h light photoperiod (3000 lux light intensity).
Results:
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14. H. Wenzler, G. Mignery, G. May, W. Park, A rapid and efficient transformation method for the production of large numbers of transgenic potato plants, Plant Sci. 63 (1989) 79-85.

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Claims (14)

1. A medium for regenerating potato plants from explants, said medium comprising:
MS salts;
one of MS vitamins or B5 vitamins;
a concentration of 30-50 mg/1 of adenine' SO4 a concentration of 18-22 g/1 of glucose;
a concentration of 18-22 g/1 of mannitol;
a concentration of 700-1500 mg/1 of MES;
a concentration of 0.03-0.05 mg/1 of gibberelic acid (GA3);
a concentration of 0.015-0.03 mg/1 of NAA;
and a concentration of 1.5-5 mg/1 of zeatin riboside;
said medium adjusted to a pH of 5.6-5.8 with 1N KOH.

2. The medium of claim 1 further comprising solidified agarose, said agarose concentration ranging from 3.8 to 4.2 g/l.

3. The medium of claim 2 wherein said agarose concentration is about 4 g/l.

4. The medium of any one of claims 1 to 3, wherein said glucose concentration is about 20 g/l.

5. The medium of any one of claims 1 to 4, wherein said mannitol concentration is about 20 g/l.

6. The medium of any one of claims 1 to 5, wherein said GA3 concentration is about 0.04 mg/l.

7. The medium of any one of claims 1 to 6, wherein said NAA concentration is about 0.02 mg/l.

8. The medium of any one of claims 1 to 7, wherein said zeatin riboside concentration is about 2 mg/l.

9. A combination for regenerating potato plants, said combination comprising a medium, and an axenic explant, said medium comprising:
MS salts;
one of MS vitamins or B5 vitamins;
a concentration of about 30-50 mg/l of adenine SO4;
a concentration of about 18-22 g/l of glucose;
a concentration of about 18-22 g/l of mannitol;
a concentration of about 700-1500 mg/l of MES;
a concentration of about 0.03-0.05 mg/l of gibberelic acid (GA3);
a concentration of about 0.015-0.03 mg/l of NAA;
a concentration of about 1.5-5 mg/l of zeatin riboside;
a concentration of about 3.8-4.2 g/l agarose;
said medium adjusted to a pH of about 5.6-5.8 with 1N KOH; and said explant comprising one of shoots, petioles and leaves.

10. The combination of claim 9, wherein said explant is a petiole.

11. A one-step regeneration protocol for potato plants, said protocol comprising placing an axenic explant in an axenic medium and culturing said axenic explant under axenic culture conditions, said axenic explant comprising one of leaves, stems and petioles, said axenic medium comprising:
MS salts;

one of MS vitamins or B5 vitamins;
a concentration of about 40 mg/1 of adenine' SO4;
a concentration of about 20 g/1 of glucose;
a concentration of about 20 g/1 of mannitol;
a concentration of about 900 mg/1 of MES;
a concentration of about 0.04 mg/1 of gibberelic acid (GA3);
a concentration of about 0.02 mg/1 of NAA;
a concentration of about 2 mg/1 of zeatin riboside;
a concentration of about 4 g/1 agarose;
said medium adjusted to a pH of about 5.7 with 1N KOH, and and said axenic culture conditions comprising a temperature ranging from 22° to 26°C
with photoperiod ranging from 14 to 18 h light and a light intensity ranging from 2500 to 3200 lux.

12. The protocol of claim11 wherein said axenic explant is a petiole.

13. A use of a regeneration medium for the one step regeneration of potato plants from an axenic explant, said regeneration medium comprising:
MS salts;
one of MS vitamins or B5 vitamins;
a concentration of about 40 mg/1 of adenine' SO4;
a concentration of about 20 g/1 of glucose;
a concentration of about 20 g/1 of mannitol;
a concentration of about 900 mg/1 of MES;
a concentration of about 0.04 mg/1 of gibberelic acid (GA3);
a concentration of about 0.02 mg/1 of NAA;
a concentration of about 2 mg/1 of zeatin riboside; and a concentration of about 4 g/1 agarose;
said medium adjusted to a pH of about 5.7 with 1N KOH.

14. The use of claim 13 further comprising transforming said axenic plant by Agrobacterium-mediated transformation.