Plant materials

2.4.1.1

PROCEDURES TO SAMPLE PLANT POPULATIONS

The same procedure as for creating a working or core collection (Harlan, 1972 loc.

cit. Spagno1etti-Zeuli and Qualset, 1993; Frankel and Brown, 1984

1

oc. cit. Spagno1etti-Zeuli

and Qualset, 1993) was applied when a representative sample of plants was necessary to

characterise populations for genetical studies. Details are presented in the corresponding

Material and Methods sections in this thesis and a brief review of different methodologies is

presented here. The objective is to minimise the cost of germplasm conservation while

ensuring

maximum

genetic diversity containing most of the alleles present in the whole

collection (Crossa et al. , 1993: Holbrook et al. , 1993; Spagno1etti-Zeuli and Qualset, 1993;

Diwan et al. , 1994).

To select a core collection from the U.S. Germplasm Collection of peanut (Arachis

hypogaea L.) consisting of 7432 accessions, two methods were used (Holbrook et al. , 1993).

When information was poor or unavailable, a random sample of 10% of the accessions was

chosen. When the information was available, the data was sorted by country of origin and

then the cluster procedure (SAS Institute, 1988) was used to sort the data and 10% of the

accessions in each cluster were randomly selected. The means and ranges for the six variables

considered were very similar for the entire collection and the core collection (Holbrook et al. ,

1993).

Diwan et al. (1994) obtained a core collection for the United States annual Medicago

Germplasm collection containing 3 159 accessions from 36 species. A SAS macro (Jacobs,

1990 loCo cit. Diwan et al. , 1994) calculated a distance matrix for each of

the

Medicago

species based on Euclidean distances between all 1 4 traits, to conduct cluster analysis using

an unweighted pair group method with arithmetic averages. Euclidean distance of 3.0 was

Literature Review

4 4

used to obtain the desired core collection size ( 15% of the accessions). Means, variances and

ranges of each trait were compared between the core collection and the main collection using

a Wilcoxon rank-sum non-parametric test (SAS Institute, 1988). Differences between means

of three or fewer traits were found significant for eight of the 36 species. Only two species

were significantly different for each trait for variances and ranges.

Crossa et

al.

(1993) suggested that a useful strategy for fonning a core collection

would be to use a stratified sampling strategy subdividing the accessions into non-overlapping

groups based on ecogeographical criteria. Classification techniques such as cluster analysis

and ordination methods such as principal components analysis have proved to be useful for

assessing genetic diversity.

Spagnoletti-Zeuli and Qualset ( 1993) evaluated five strategies for obtaining a core

collection of 500 accessions from a collection of 3000

acces

sions of durum wheat (Triticum

turgidum L. d

urum

group). The strategies were ( 1) random-sampling without replacement;

(2) random-systematic by chronology - sample every fourth accession in the order in which

the accessions were accepted by the gene

bank;

(3) random-stratified by geographical origin

and frequency-selecting at random 16% of the accessions of each country; (4) random­

stratified by log frequency of accessions by geographical origin - same as (3) but countries

with large number of accessions contributed proportionally fewer accessions to the core

collection and the opposite for countries with few accessions; and (5) random-stratified by

canonical variables - based on the concept that pre-existing information is available; the first

three canonical variables were plotted and about 10% were randomly selected.

The fIrst three strategies produced representative samples, but strategies four and fIve

produced the desired effect of increasing frequencies from less-represented countries of origin

for several traits. The fifth strategy was the best and was effective in increasing the phenotypic

variances in the sample for most characters, due mostly to the increase of the less-frequent

accessions and a decrease in the most-frequent ones, thus flattening the frequency

distnbution.

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4 5

If necessary information is not available for the :fifth strategy the others are adequate to each level of information available.

2.4.1.2

CLONING PROCEDURE

Identical copies of the same genotype of Red clover plants can be produced by cloning. Such clones can be useful in testing some issues discussed here.

Scerbakova

( 1 936)

compared the rooting and development of cuttings taken from

the

upper-stem, middle-stem and basal-stem The upper cuttings rooted better than the middle ones, but both regenerated plants with only a single stem Basal cuttings had the poorest rooting but they regenerated plants with normal stem numbers. Cuttings from

the

crown were also tried, and they showed the best rooting. Rooting of all

the

cuttings was better in sand

(83.33%)

than in soil

(66.26%)

or in water

(48.33%).

Hanson

( 1 950)

found that stage of growth was important and that cuttings from actively growing vegetative plants were easier to root than cuttings from plants in the reproductive stage. He also found that temperatures between

20°C

and

30°C

were optimum for rooting. The influence of the length of the internode below the last node was also studied by the author, who concluded that internode length should be shorter than

1 .5

cm to produce best results.

Barrales and Ludwig

(1952)

in their studies of photoperiodism found that with a day­ length of eight hours there is a gradual decrease in stem elongation and an increase in crown bud formation. Their cloning method was to cut vertically, to include at least one bud and a portion of the crown in each cutting. To reduce Wilting, the plants were kept in a cool environment with

diffuse

light and all the old leaves were taken from the cuttings. The method of stem cuttings (upper cuttings, middle cuttings and basal cuttings) was tried by the authors. Two nodes were included in each cutting and the upper leaf was left attached to the propagule. Two honnones (indol butyric and alpha naphthyl acetic acids) were applied to the

Literature Review

4 6

lower end of the cuttings to promote rooting but they were ineffective in the majority of

cases. The same environmental conditions as those applied to crown cuttings were

used.

Individual plants were found to differ in their response to clonal propagation. Crown cuttings

were the best, with 100% success in some plants, followed by basal cuttings.

Cumming and Steppler (1961) defined five possible types of propagules. Upper or tip

cuttings, middle cuttings, basal cuttings, crown cuttings and leaf-bud propagules.

The

latter

was considered by the authors to be the best type, and to show a close similarity to seedlings

in growth and development. Cumming and Steppler ( 1961) suggested that pre-treatment of

the intact plants with a short day-length environment and/or to water them with TIBA (2,3,5-

triiodobenzoic acid) in the solution could increase the rate of rooted cuttings. Treatment with

IAA (indo1acetic acid) and increase in day length during the rooting stage were

recommended. Overhead misting systems enabled the propagules to remain turgid and

reduced wilting problems, without the need of shade or a cool environment.

Mirzaie-Nodoushan and Gordon (1993)

used

the technique developed by Barrales

and Ludwig (1952) for stem cuttings, but with the improvement suggested by Cumming and

Steppler (1961) of overhead misting to reduce wilting instead of a cool environment and

shade. They concluded that cuttings of different portions of the plant and genotypes produced

different results, not allowing a generalisation. The authors recommended the use of clones

from the same portion of the plant to reduce possible variability in genetical studies.

The

percentage of rooted cuttings varied from 55% to 85% depending on the genotype.

In document Quantitative genetics of sheep preference in red clover (Trifolium pratense L ) under spaced plant and sward conditions : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Ph D ) in Pasture Breeding, Depa (Page 76-79)