Gestión de los Planes

The following chapters of this section evaluated the genetic stability of the asparagus genome throughout somatic embryogenic culture. Further details of the objectives and experimental approach for this section were outlined in Chapter 1 .

These objectives were achieved by analysing long-term embryogenic calli maintained on medium containing PGRs for 1 Y2 years, long-term embryogenic cells maintained as suspensions on PGR-free media for 1 year, and regenerated plantlets. Analysis was made using RAPDs and cytological techniques. The potential use of extra-cellular proteins to reveal genetic variation was also investigated using protein gel electrophoresis techniques. The merits and limitations of these techniques have been outlined in previous sections of this chapter.

CHAPTER FIVE

Detection of variation among and within asparagus hybrids using random amplified DNA (RAPD) markers

Abstract

The reliability of random amplified polymorphic DNA (RAPD) techniques to amplify polymorphisms in the asparagus (Asparagus officinalis L.) genome was investigated. DNA fragments generated by l O-base primers were separated on 1 .5% agarose or 8% polyacrylamide gels, and the sensitivity of ethidium bromide and silver staining of amplified DNA products analysed on these gels was compared. Resolution of DNA bands on polyacrylamide gels was superior to that on agarose gels. Silver staining was more sensitive than ethidium bromide staining. The gel type used to separate DNA bands, and the staining technique used influenced the number of bands visualised for each DNA profile generated. The six asparagus cultivirs used were distinguished by unique banding patterns generated by each primer, OPC- I 2, for example, generating polymorphic markers unique to three of the cultivars investigated, ASP (500, 400, and 300 bp), TV (700 bp), and (PC 550 bp). Clear differences were noted in DNA profiles generated for samples of seed established cultivars: SN_7 ( 1 300 bp); SN_13 ( 1900, 1 800, 1700, 1 100, and 1000 bp); UC_5 (1 800, 1 500, and 1400 bp); UC_6 (2000, 1050, and 600 bp); and UC_I I ( 1 600 bp). The investigation indicates that RAPD markers can be used to characterise asparagus cultivars, and that the technique is sensitive enough to reveal differences within seed-raised commercial cultivars. RAPD technology has the potential to detect somaclonal variation occurring during micropropagation.

Keywords

Asparagus officinaiis, micropropagation, primers, RAPDs, ethidium bromide, silver

stain, agarose, polyacrylamide

A manuscript based on this experiment has been accepted for publication by New Zealand Journal for Crop and Horticultural Science.

1 05

5.1 EXPERIMENT 4

5.1.1 INTRODUCTION

Commercial micropropagation of asparagus clones requires regenerated plantlets to be genetically uniform. For this reason, it is necessary to establish reliable techniques to determine their genetic stability as the effect of genetic variation on yield within a clone has yet to be established. If in vitro techniques promote genetic variation, it is important to qualify some of the factors contributing to variation, and to quantify the extent of this variation. In the past, attention has been focused on the use of morphological, biochemical, and cytological techniques to determine the frequency of somatic mutations occurring during micropropagation (Heinz & Mee 1 97 1 ; Patel & Thorpe 1 987 ; Gonzalez-Castanon & Carbojal-Carcedo 1 996). However, both morphological and biochemical techniques are influenced by epigenetic effects and, as with cytological analysis, often fail to detect subtle genetic changes occurring at the molecular level that could have a significant impact on crop quality and yield. This is particularly important for perennial crops like asparagus that may be productive for 10-20 years.

Recently, molecular techniques such as restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNAs (RAPDs) have been used to characterise genomic DNA (Welsh & McClelland 1 990; Williams et al. 1 990; Xiao & Christie 1 994), and to detect somatic mutants in some plant species (Kaemmer et al.

1 992; Deng et at. 1 995). Analytical molecular techniques provide a methodology that allows evaluation of the plant genome independent of environmental factors, physiological state, or developmental regulation. RAPD techniques have the added advantage over RFLPs of being relatively simple and quick to perform requiring only small quantities of DNA for analysis. In addition, no prior knowledge of genomic DNA sequence is required for RAPD analysis (Williams et al. 1 990). As a consequence, RAPDs have been used to identify and characterise closely related plant species and cultivars (Munthali et al. 1 992; Torres et al. 1 993; Yang & Quiros 1 993).

Before RAPD techniques can be used either for characterisation of closely related asparagus cultivars or to detect somaclonal variation occurring during in vitro

Chapter 5 RAPD analysis of explants

production of asparagus clones, the repeatability and sensitivity of the RAPD approach must be evaluated. This report investigated: the reliability of RAPD DNA fingerprints generated by selected 1 0-base primers; the ability of RAPD techniques to distinguish among asparagus cultivars; and, the sensitivity of the technique for revealing differences within a seed-raised commercial cultivar.

5.1.2 MATERIALS AND METHODS

5.1.2.1 Plant material

Three of the asparagus cultivars Pacifica (PC), UC 1 57 (UC), and Syn 4 (SN) used were

maintained at the greenhouse units at Massey University. Both 'UC' and 'SN' were established from seed, whereas 'PC' was established from clonal material. Cultivars

Turoa (TU), Aspiring (ASP) and Karapiro (KP) were clonal material obtained and

maintained as tissue-cultured liners from Aspara Pacific Ltd, Christchurch, New Zealand. Plant material used in this investigation is listed in Table 5. 1 .

5.1.2.2 DNA extraction

Before DNA extraction, samples of actively growing young fern or spears were dried for a minimum of one week in sealed plastic bags containing silica gel. The procedure for DNA extraction was a modification of Doyle & Doyle' s ( 1 990) cetyltrimethylammonium bromide (CT AB) method and the minipreparation technique

of Stewart & Via ( 1 993) (P Lockhart pers. comm. 1996). The isolation buffer (2% CTAB (BDH), 1 .5 M NaCI, 1% polyvinylpyrolidone 44 000 (PVP-44) (BDH), 20 mM

EDTA, 100 mM Tris-HCI (pH 8.0)) was preheated to 60°C. Milligram amounts (20-50 mg) of dried plant tissue were ground in a 1 .5 ml eppendorf tube in liquid air. Hot isolation buffer (0.7 ml) was added and the sample incubated at 60°C for 30-60 min. An equal volume of chloroform was added and the sample centrifuged briefly at 10 000 rpm. The aqueous phase was transferred to a clean tube and two thirds volume of isopropanol added. The white DNA precipitate was recovered by spooling, washed three or four times in 80% ethanol, dried, and resuspended in 50 III sterile water

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(Barnstead® nanopure) . The quantity of DNA extracted was estimated by electrophoresis and separation of the sample DNA and known concentrations of lambda DNA (Pharmacia Biotech) on a 0.8% agarose gel, and comparing the intensity of bands visualised after staining with ethidium bromide (Sambrook et aI. 1 989b).

Table 5.1 Asparagus (Asparagus officinalis L.)

cultivars evaluated by random amplified polymorphic DNA Cultivar Aspiring Karapiro Turoa Pacifica Pacifica Pacifica Syn 4 Syn 4 VCl S7 VC157 VC1 57 5.1.2.3 RAPD procedure Sample i.d ASP KP TO PC9 PC_12 PC_16 SN_7 SN_13 VC5 VC_6 VC 1 1 Propagation Sex method Male clone Male clone Male clone Female clone Female clone Female clone Female seed Female seed Female seed Female seed Female seed

Primers used for RAPD analysis were obtained from Operon l O-mer Kit C (Operon Technologies). A modification of the procedure of Williams et aI. ( 1 990) was used. RAPD reactions were performed in a total volume of 25

I.d

polymerase (Gibco-BRL), 2 mM MgCh and IX PCR buffer (supplied with Taq polymerase), 5 pmole primer, 1 00 /lM of DNA polymerisation mix (Pharmacia Biotech) (containing dATP, dCTP, dGTP, and dTTP), and 25 ng template DNA. The reaction mixture was overlaid with one drop (50 /l1) of mineral oil. For control reactions, template DNA was excluded from the reaction mixture. To avoid contamination of DNA from other organisms, all PCR reactions were prepared in a laminar flow cabinet. Polymerase chain reaction (PCR) cycling conditions were as follows : 1 cycle of 5 min at 94°C followed by 40 cycles of 1 min at 94°C, 1 min at 36°C, 2 min at 72°C and a final time delay cycle of 5 min at 72°C followed by an optional soak period at 4°C. This soak period was required for PCR reactions run overnight. PCR reactions were performed in a Perkin Elmer Cetus DNA Thermal Cycler 480. Amplified products were separated by

Chapter 5 RAPD analysis of explants

electrophoresis in a 1 .5% agarose gel. A Tris-borate EDT A (TBE) buffer system was used. To avoid over-heating of electrophoresis buffer, gels were run at 4°C.

5.1.2.4 Improved resolution of amplified bands

For increased resolution, amplified products were separated on an 8% polyacrylamide gel and stained with ethidium bromide. Ethidium bromide stained gels were de stained in water for 20-30 min as recommended by Andrews ( 1 986), and then silver stained. The silver staining procedure was a modification of Igloi's ( 1 983) modified Sammons'

procedure. The modifications consisted of shorter incubation times at each step (H F Neilson pers. comm. 1996). Gels were initially washed twice for 5 min in ethanol and acetic acid, stained in AgN03 for 1 5 min, washed in running distilled water for 1 min, developed for 10 min, and fixed for 5 min. The procedure was completed in 36 min instead of the 1 90 min recommended by Igloi ( 1 983).

5.1.2.5 RAPD analysis of 'Syn4' and 'Ue157' seedlings

The DNA profiles of 1 00 seedlings of cultivar Syn4 and UC 1 57 were analysed to determine the degree of intra-cultivar variability within cultivars. DNA extraction and amplification protocols were the same as used previously.

5.1.3 RESULTS

5.1.3.1 Screening of explants with to-base primers

Initial screening of explants from each cultivar was performed with twenty l O-base primers. Analysis was based on the visual resolution of DNA bands on agarose gels. Criteria for selection were based on the ability of each primer to produce 5- 10 clearly discernible bands for evaluation and comparison, as well as being able to produce unique fingerprints for several of the cultivars under investigation. Based on these criteria, the following 10 primers were selected from Operon Kit C: OPC-O I , -02, -07, - 08, -09, - 1 0, - 1 1 , - 1 2, - 1 3, and - 14. The number of amplified bands produced by each

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primer, for all cultivars, ranged from 5 to 15, with an average of 1 1 bands per primer. Amplified DNA fragments were in the range of 400-2400 bp. The DNA profiles generated for each cultivar by 20 primers are found in Appendix Table 5.0.

5.1.3.2 Polymorphisms produced by different primers

The DNA fingerprints produced by 10 primers for cultivars PC_9 and SN_7 are shown in Plate 5 . 1 each primer investigated producing a distinct pattern of amplified bands. The difference in fingerprints generated for each cultivar is also evident in Plate 5 . 1 .

5.1.3.3 Improved resolution of amplified products

Separation of amplified products was better on 8% polyacrylamide gel (Plate 5.2a) than on 1 .5% agarose gel (Plate 5.2b). Staining with ethidium bromide did not reveal additional more intensely stained bands than in agarose. However, very high molecular weight bands (greater than the largest band on the marker), and very low molecular weight bands of 100 bp were evident on acrylamide gels. These bands were not visible on similar stained agarose gels (Plate 5.2b).

The gel in Plate 5.2a was de stained and then silver stained. The increased sensitivity of this staining technique is clearly shown in Plate 5.2c. The intensity of the silver stained bands, and the number of bands evident, was greater than when stained with ethidium bromide. An average of 23 amplified bands were generated by OPC- 1 2 when silver stained, more than twice the average number of bands revealed on agarose and acrylamide gels stained with ethidium bromide. Thirteen heavily stained bands were revealed on this gel, and these bands could be useful markers for further investigations. The results with OPC- 1 2 were typical of other primers used in this study (data not shown).

Chapter 5 RAPD analysis of explants 1 10