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Induction of qualitative marker traits(s), via DNA macroinjections in cotton and its characterization with RAPD markers.

Cotton due to its economic importance has long been subjected to extensive research in Pakistan. Breeders are always striving to change the genetic constitution of the plant for improving both its yield and fibre quality under diverse climatic conditions of the country. Since the variability for desirable traits in the existing cultivars is scanty. Therefore, various methods like, hybridization, backcrossing and ovulo-embryo culture, etc. are being used to generate variability by incorporating the desirable genes into G. hirsutum from exotic sources (Stewart 1992). These approaches are not only time consuming but also are confronted with the problems, due to differences in ploidy level, interspecific barriers, and undesirable gene linkage (Nishiyama and Yabuno, 1978). Induced mutations have also been used to create genetic variability in many crop plants, and in recent years a large number of early, high yielding, disease and insect resistant varieties of various crop plants, e.g. wheat, barley, rice, cotton, etc., have been developed and released in different countries of the World (Micke, et al, 1987).

However, different transformation approaches (Agrobacterium mediated transformation and electroporation of DNA for gene transfer) are also being used to incorporate desirable genes into crop plants more quickly. Agrobacterium mediated transformation is the most commonly used method for gene transfer in plants (Horsch, et al. 1984). Electroporation of DNA for gene transfer using protoplasts has also been used successfully for the production of transgenic plants (Fromm et al. 1985 and Shillito, et al. 1985). Some other in vitro transformation methods, e.g. DNA microinjections in tobacco (Crossway, et al. 1986) and rye (De La Pena, et al. 1987) and particle bombardment in cotton (Finer and McMullen, 1990) have been used. These transformation methods require callus culture to regenerate whole plant from transformed protoplast or cell. Since the ability of cotton genotypes to regenerate from a cell into a whole plant, through callus tissue is genotype-specific (Gawal and Robacker1990 and Trolinder and Xhixian, 1989), therefore it is difficult to regenerate, the whole fertile plant that expresses inserted genes, which can be passed on to its progeny. On the other hand DNA-mediated embryo transformation is a straightforward in vivo approach in which exogenous DNA solution is injected into the plant's reproductive structure during fertilization process. The injected DNA solution thus transforms the developing embryos during zygotic cell division and this transformation is heritable. Positive (in vivo) transformations in cotton have been reported (Aslam et al. 1995, 1997, 1998, and Zhou, et al. 1982, 1983), but still lack molecular confirmation/explanation.

The research studies reported herein were carried out under an IAEA research contract 8 142/RB, titled, "Development of improved germplasm of cotton through radiation and DNA- mediated embryo transformation technique." with the objectives to incorporate qualitative marker traits (petal spot, pollen colour) of the donor parents into G. hirsutum and their subsequent characterization with morphological and RAPD markers (Williams et al. 1990).

Materials and methods

A tetraploid species of cotton, G. hirsutum (2n=4x=52) var. NIAB-78, was used as recipient while a diploid species of cotton, G. arboreum (2n=2x=26) var. Ravi, and a tetraploid long staple species, G. barbadense (2n = 4x = 52), were used as donors. G. barbadense L. is the most popular species for its superior quality fibre, while G. arboreum L. is best reported for high fibre strength, resistance to all prevalent insects and known diseases (Stanton, et al. 1994 and Stewart 1992). Both of which were clearly distinguishable morphologically (leaf shape, flower colour, pollen colour, boll shape etc.) from the recipient. Both donors were grown from selfed seed under controlled conditions. The DNA was extracted from young leaf tissue of the donors and DNA solutions for injections were prepared as previously reported (Aslam et al., 1998).

At flowering, 50 healthy plants of the recipient raised from selfed seed were chosen. The self-fertilized flower/ovaries of these were injected via microsyringe with irradiated and non-irradiated donor DNA solutions through the axial placenta 24 hours post self-pollination. For each treatment 10-microliter DNA solution was used and the injected flower/ovaries of the recipient were protected from foreign contamination. Matured bolls from the treated flower/ovaries were collected, ginned and [D.sub.0] generation seed was developed. The [D.sub.0] seed thus developed was evaluated in [D.sub.1] and [D.sub.2] generations (Aslam, et al. 1998). The [D.sub.1] and [D.sub.2] populations were exposed to CLCuV disease under natural high disease infestation during the consecutive years, using spreader rows of a highly susceptible cultivar S-12 to encourage uniform inoculation. The highly susceptible cultivar S-12 received 100 per cent disease infestation and this disease intensity was measured as described by Siddig (1968). The [D.sub.2] generation plants expressing donor parents qualitative traits were grown as the [D.sub.3]generation for evaluation.

Young leaves of different [D.sub.2]-derived [D.sub.3] transformed plants along with their parents were used for RAPD analysis. The young leaves collected in liquid nitrogen were used for DNA extraction and PCR analysis as reported earlier (Aslam, et al. 1998). After RNase treatment, the DNA concentration was measured by DyNA Quant 200 Fluorometer. The DNA was diluted in sterilized distilled water to a concentration of 12.5 ng/ml for use in PCR reactions for RAPD analysis.

Random decamer primers (Operon Technologies Inc., Alameda, Calif., USA) were dissolved in sterilized distilled water at a concentration of 15 ng/ml. Thirty two primers belonging to Operon kits; OPM (20 primers) and OPB (12 primers) were used for PCR amplifications. A reaction mixture of 25 ml containing 10 mM Tris-HCl (pH 8.3 at 25[degrees]C), 50 mM KCl, 3 mM Mg[Cl.sub.2], 0.1 mM each of dATP, dGTP, dTTP and dCTP, one unit of Taq DNA polymerase (Perkin Elmer, Norwalk, Conn.), 0.001% gelatin (Sigma, St-Louis, Mo.), 25 ng of template DNA and 30 ng of primer was prepared and overlaid with two drops of mineral oil in order to avoid evaporation. The amplifications were carried out in a Perkin Elmer Thermal Cycler 480, programmed for a first denaturation step of 5 minutes at 94[degrees]C followed by 40 cycles of 94[degrees]C for 1 minute, 36[degrees]C for 1 minute and 72[degrees]C for 2 minutes. After the completion of 40 cycles, the reactions were kept at 72[degrees]C for 7 minutes and then held at 4[degrees]C until the tubes were removed. Amplified products were separated on a 1.2% agarose gel with ethidium bromide in the gel, using 0.5 x Tris Borate EDTA (TBE) buffer.

Results and Discussion

Results obtained with macroinjection of DNA extracted from G. arboreum indicated that the [D.sub.1] population obtained from irradiated (2.5Gy) donor DNA treatments exhibited higher percentage of transformations than the [D.sub.1] population obtained from the treatments with non-irradiated donor DNA (Table 1). Some of the transformed plants exhibited resistance to CLCuV disease and also showed changes in boll size/weight, boll shape, plant type, hairiness, gossypol pigments, etc. The [D.sub.1] generation plants had more hairiness, more monopodial branches and good boll formation. But no changes were observed for flower colour and petal spot in the [D.sub.1] generation. The [D.sub.2] generation results indicated the persistency in the changes observed during [D.sub.1] generation (Table 2). Moreover, some of the [D.sub.2] progenies manifested qualitative traits, e.g. petal spot, pollen colour, of the donor G. arboreum. Moreover the CLCuV resistant plant progenies showed segregation, ratio of 3:1 for resistant and susceptible genotypes, indicating that CLCuV resistance behaved as a monogenic and dominant (RR) trait (Aslam et al. 1999 and Mahbub 1997). Moreover, plant progenies grown from [D.sub.2] plants expressing qualitative traits incorporated from the donor, such as petal spot and yellow pollen colour, showed segregation of these traits in [D.sub.3] generation. The segregation patterns reflected Mendelian inheritance giving 1:2:1: ratio for incompletely dominant traits (petal spot), and 3:1 for dominant traits like pollen colour. (Table 3).

Results obtained with macroinjection of DNA extracted from G. barbadens into G. hirsutum revealed that some of [D.sub.1] plants had changes for plant type, boll type/size, leaf shape, plant vigour, etc. The spectrum of such changes for donor parent traits was higher with the irradiated DNA treatments (Table 4). The [D.sub.2] plant progeny rows showed consistency in the changes noted during the [D.sub.1] generation. Moreover some of the [D.sub.2] progenies expressed changes for qualitative traits (typical of the donor parent G. barbadense), such as red petal spot, pollen colour and flower colour (Table 5). Various transformed plants of the [D.sub.2] generation having qualitative trait changes were evaluated in [D.sub.3] generation. The [D.sub.2] plants having changes in qualitative traits, such as red flower petal spot and yellow pollen colour, showed Mendelian segregation for these characters in [D.sub.3] generation (Table 6).

Thirty-two primers were used to amplify gnomic DNA of different [D.sub.2]-derived [D.sub.3] transformed cotton plants and their respective parents for PCR analysis (Aslam et al. 1999). PCR with the primer OPM-04 resulted in polymorphic products among the samples. A band of 750 bp, present in G. arboreum (lane 12) was also present in a transformed progeny, (lane 18) but was absent in all other samples (Fig. 1). An other primer, OPM-11, also resulted in a G. arboreum-derived polymorphic band of 1 kb present in the same plant (Fig. 2) that was not detected in any other samples. A third primer, OPM-19, revealed a 750 bp band, in G arboreum (lane 12) and a different progeny (lane 14) that was absent in all other samples (Fig. 3). Farooq et al. used RAPD markers for identification of wheat genotype (Farooq et al. 1994), for identification of cultivated and wild rice species (Farooq et al. 1995) and to see the inheritance of RAPD markers in F1 interspecific hybrids of rice (Farooq et al. 1996). In the study of inheritance of RAPD markers ((Farooq et al. 1996) particularly observed that appearance of RAPD markers specific for donor parents is dependent upon the choice of primers. They have suggested that in order to see how many DNA segments specific for the donor parents can be detected in the recipient parents, it is necessary to use a large number of random primers of varied nature. In the present study we have used limited primers from only two kits (OPM & OPB) which detected only three fragments specific for G. arboreum in two plants. Unless these fragments are tagged with the character of interest that were to be transformed from the donor parent, it would be difficult to say that that the transformation obtained through morphological observation is potentially appeared due the presence of their fragments However their presence in the [D.sub.3] progenies did indicate that it is possible to transform plants through DNA microinjection. However fort detection of maximum number of DNA fragments specific for donor parents in the recipient it is advisable to use as many primers as possible.




The results of [D.sub.1] and [D.sub.2] generations originating from G. hirsutum x G. arboreum and G. hirsutum x G. barbadense DNA treatments concluded the enhanced introgression of donors gene(s) into the recipient, where the DNA was irradiated at low doses (2.5 Gy) of gamma rays before injections (Kohler et al. 1989). Some of the transformants originating from G. arboreum DNA injections into G. hirsutum were resistant to CLCuV disease. Moreover, introgression of some of the qualitative marker traits, such as red petal spot, yellow pollen colour, etc., from the G. barbadense and G. arboreum donors into G. hirsutum confirmed the previous findings

(Aslam et al. 1997).

Observations on phenotypic changes in progeny following injection of G. hirsutum with foreign DNA indicated that at least some of the DNA was incorporated into the genome. Similar results for gene(s) incorporation in barley (Soyfer 1980) in tobacco (Crossway et al. 1986) in cotton (Finer and McMullen, 1990) using different biotechnological approaches have been reported. With a limited number of primers in PCR reactions, we confirmed at the molecular level that G. arboreum DNA was incorporated. However, with this same primer set we could not confirm incorporation of G. barbadense DNA probably due to the low level DNA polymorphism.


Acknowledgements are due to I.A.E.A., Vienna, Austria for partly financing these studies under research contract 8142/RB. Thanks are due to Director NIAB, for encouragement and help and to former Director General NIBGE/ NIAB, Dr. K. A. Malik and to Director/ Scientists of NIBGE for performing the RAPD analyses.


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(1) M. Aslam, (3) M. Ashfaq, (2) Y. Zafar, (3) Sami ul Allah and (3) Muhammad Sajjad

(1) Nuclear Institute For Agriculture & Biology, P O Box 128, Jhang Road, Faisalabad Pakistan.

(2) National Institute For Biotechnology & Genetic Engineering, P.O. Box 577, Jhang Road, Faisalabad Pakistan.

(3) Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan

Corresponding Author: Muhammad Sajjad, Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan

Table 1: Phenotypic evaluation of [D.sub.1] generation from the
injections of G.arboreum DNA into flower-ovaries of G.hirsutum
during 1995-96.

                         Plants    Changed
Treatments               studied   plants
                         (No.)     (No.)

G.hirsutumxG.arboreum    9         1
DNA (0 Gy)

G.hirsutumxG.arboreum    32        7
DNA (2.5 Gy)

G.hirsutum (R) NIAB-78   20        --
G.arboreum (D)           20        --
S-12 (S)                 20        --

Treatments               Type of change

G.hirsutumxG.arboreum    i). The transformed plants showed
DNA (0 Gy)               changes for boll shape/size, plant type,

G.hirsutumxG.arboreum    ii). Some of these were found resistant
DNA (2.5 Gy)             to CLCuV.

                         iii). No marker/qualitative trait changes
                         were noticed.

G.hirsutum (R) NIAB-78
G.arboreum (D)
S-12 (S)

CLCuV Rating Scale 0-9 i.e. 0 = Immune, 9 = Highly susceptible,
R = Recipient, D = Donor, S = Standard Highly susceptible

Table 2: Phenotypic evaluation of [D.sub.2] generation plant progenies
of G. hirsutum x G. arboreum DNA injections during 1996-97.

                                   Plant studied (No)
Name of progeny

                                   Total   Resis.   Suscep.
DNA (2.5 Gy)
G. hirsutum xG. arboreum    -1     50      37       13
"                           -3     51      39       12
"                           -4     58      43       15
"                           -5     44      32       12
"                           -6     51      38       13
"                           -8     46      34       12
"                           -9     45      34       11
DNA (0 Gy)
G. hirsutumxG. arboreum     -8     45      33       12
G. hirsutum (R) NIAB-78            47               47
S-12 (Standard)                    49               49

Name of progeny                    (approx.)         Plants with
                                   Resis.            Markers traits
                                   / Suscep.         (No.)
DNA (2.5 Gy)
G. hirsutum xG. arboreum    -1     (3:1)             --
"                           -3     (3:1)             3*
"                           -4     (3:1)             --
"                           -5     (3:1)             2*
"                           -6     (3:1)             3*
"                           -8     (3:1)             --
"                           -9     (3:1)             --
DNA (0 Gy)
G. hirsutumxG. arboreum     -8     (3:1)             --
G. hirsutum (R) NIAB-78            all susceptible   --
S-12 (Standard)                    all susceptible   --

CLCuV Rating Scale: 0-9 i.e. 0 = Immune, 9 = Highly susceptible

* Plants had petal spot and yellow pollen colour, etc.

Table 3: Evaluation of D3 progenies of G. hirsutum x G.arboreum *
DNA injections for qualitative traits during 1997-98.

Name of Progeny                               Number of plants with

                                  Y. Pollen   C. pollen   P. Spot
G. hirsutumxG. Arboreum    -3-1   --          --          12
"                          -2     28          10          --
"                          -3     23          7           --
"                          -5-1   54          19          --
"                          -2     --          --          27
                           -6-1   71          25          --
"                          -2     38          12          --
"                          -3     40          14          --

Name of Progeny
                                  Number of plants with

                                  NPS     Others     Total
G. hirsutumxG. Arboreum    -3-1   14      24 LPS *   50
"                          -2     --      --         38
"                          -3     --      --         30
"                          -5-1   --      1.LPS.     74
"                          -2     21      --         48
                           -6-1   --      1.LPS.     97
"                          -2     --      --         50
"                          -3     --      --         54

* LPS = light petal spot

C = Cream,

P = Petal,

NPS = Non petal spot

Y = Yellow

Table 4: Phenotypic evaluation of D1 generation from injections
of G. barbadense DNA into flower /ovaries of G. hirsutum during 1995-96.

                          Plants    Changed
                          studied   plants
Treatments                (No.)     (No.)

G.hirsutumxG.barbadense   31        3
DNA (0 Gy)

G.hirsutumxG.barbadense   40        6
DNA (2.5 Gy)

Recipient * NIAB-78       22        --
Donor **                  19        --

Treatments                General Observations

G.hirsutumxG.barbadense   The changed plants:
DNA (0 Gy)                i). Were faster in growth.

G.hirsutumxG.barbadense   ii). Had larger leaves conical bolls and
DNA (2.5 Gy)              long petioles.

Recipient * NIAB-78       iii). Had larger to medium flower with
Donor **                  bigger calyx.

                          iv). Had no evidence of marker traits
                          i.e. petal spot, pollen colour.

* G.hirsutum

** G.barbadense

Table 5: Evaluation of D2 generation of G.hirsutumxG.barbadense
DNA injections during 1996-97.

                                  Quality   Characters
                                  length    fineness    strength
Name of progeny                   (mm)      <>g/in      (TPPSI)

G.hirsutum x G.barbadense   -1    28.5      3.8         99
(DNA, 0Gy)
"      " "                  -5    28.5      3.8         98
"      " "                  -9    29.0      3.9         99
G.hirsutumxG.barbadense     -7    30.0      3.6         99
(DNA (2.5 Gy)
"      " "                  -9    29.7      3.7         100
"      " "                  -20   29.3      3.8         101
"      " "                  -29   29.2      3.5         98
"      " "                  -30   29.1      3.7         97
G.hirsutum (R) NIAB-78            27.3      4.5         90

                                  Plants with
                                  marker traits
Name of progeny                   (No.).

G.hirsutum x G.barbadense   -1    1 yellow pollen
(DNA, 0Gy)
"      " "                  -5    --
"      " "                  -9    --
G.hirsutumxG.barbadense     -7    1 petal spot
(DNA (2.5 Gy)
"      " "                  -9    1 yellow pollen
"      " "                  -20   1 petal spot
"      " "                  -29   --
"      " "                  -30   --
G.hirsutum (R) NIAB-78

Note: Besides other [D.sub.1] phenotypic changes the D2 plants
also had transformations for i) Red flower petal spot, ii) and
Yellow pollen colour.

Table 6: Evaluation of D3 progenies of G.hirsutum x G.barbadense
DNA treatments for qualitative traits during 1997-98.

Name of Progeny                     Number of plants with

                                Y. Pollen   C. pollen   P. Spot

G.hirsutumxG.barbadense   -1    29          10          1 DPS
(0 Gy)
G.hirsutumxG.barbadense   -7    --          --          8
(2.5 Gy)
"                         -9    32          13          1 LPS
"                         -20   --          --          11
G.hirsutum (R) NIAB-78          --          --          --

Name of Progeny                 Number of plants with

                                NPS    Others    Total

G.hirsutumxG.barbadense   -1    --     --        40
(0 Gy)
G.hirsutumxG.barbadense   -7    10     21.LPS    39
(2.5 Gy)
"                         -9           1 Y.Pt.   47
"                         -20   13     25 LPS    49
G.hirsutum (R) NIAB-78          --     --        20

* LPS = light petal spot C = Cream, P = Petal, NPS = Non petal spot
Y = Yellow PS = Petal spot Y.Pt. = Yellow petal
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Title Annotation:Original Articles
Author:Aslam, M.; Ashfaq, M.; Zafar, Y.; Allah, Sami ul; Sajjad, Muhammad
Publication:American-Eurasian Journal of Sustainable Agriculture
Article Type:Report
Geographic Code:6NIGR
Date:Dec 1, 2009
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