DESC:TECHNICAL FIELD

The present disclosure generally relates to genetic modifications and crop improvement through chemical mutagenesis and more particularly relates to a pure line diploid plant or shoots thereof produced from diploid seeds of Guizotia abyssinica under in vitro condition and a chemical mutagenesis method for producing tetraploid plants of guizotia abyssinica cass. The disclosure furthermore relates to a tetraploid plant produced by said method. The produced tetraploid plant has increased ploidy status (doubled chromosomes); increase chlorophyll content (darker green color), and in floral characteristics such as having more large-sized sepals, increased diameter of capitulum, and reduced number of capitulum per plant resulting in more seed yield, and subsequent improvement in the productivity and stress tolerance ability.
DESCRIPTION OF THE RELATED ART
The Food and Agriculture Organization defines food security as “situation that exists when all people, at all times, have physical, social, and economic access to
sufficient, safe, and nutritious food”. During the last two decades, India has been

facing problem of shortage and raise of edible oil price and consumption.

Nearly 85% of India’s oilseeds production is from rain fed areas and hence there are wide fluctuations in production due to monsoon. Oilseeds are grown in nutrient poor soil and limited land area for cultivation. Progress in the evolution and introduction
of high yielding hybrid varieties is poor as compared with cereals and cotton.

Owing to these factors, the yield per hector is very low.

To satisfy the need of edible oil of overgrowing population, there is need to increase the land under cultivation as well as to increase the productivity of oil seed crops.

SUMMARY OF THE DISCLOSURE
The present disclosure relates to a pure line diploid plant or shoots thereof produced from diploid seeds of Guizotia abyssinica under in vitro condition.
The present disclosure further provides a method of producing a tetraploid plant, said method comprising the steps of:
i) growing a pure line diploid plant in vitro of said plant, wherein the plant is an edible oil seed crop belonging to family Asteraceae but not limited to Guizotia abyssinica;
ii) applying an anti-mitotic agent to apical portion of in vitro raised shoots of the diploid plant;
iii) treating the diploid plant of said plant with sterile distilled water for 10 minutes;
iv) inoculating said treated diploid plant on nutrient medium;
v) observing and selecting in vitro raised tetraploid shoots; inducing roots in the in vitro raised selected tetraploid shoots;
vi) assessing the chromosome number in roots and tetraploidy level in leaf by flow cytometric method of the selected tetraploid plantlets;
vii) proliferating and multiplying said selected tetraploid plant; and
viii) assessing the chromosome stability in future generations of said tetraploid plants produced.
The present disclosure furthermore relates to a tetraploid plant produced by the method as defined above.
.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments herein will be better understood from the following detailed description with reference to the drawings, in which:
FIG. 1 is a flow diagram 100 illustrating a method for producing tetraploid plants of
Guizotia abyssinica Cass according to an embodiment herein; FIG. 1A is an image of the method of producing tetraploid plants with the help of in vitro raised diploid shoots; inverted position of diploid shoot allows treatment of colchicine solution incorporated in MS medium containg BAP to apical portion of the shoot; the treated apical portion allowed to grow and proliferate as a tetraploid shoot on MS + BAP and IAA medium.

FIG. 2 illustrates a graphical representation showing leaf cell nuclear DNA content of said diploid and tetraploid plants of the third vegetative generation produced by the method in accordance with the preferred embodiment of the present invention, wherein histograms represent double the nuclear DNA content.
FIG. 3 illustrates a graphical representation for number of chromosome in leaf derived roots of diploid and tetraploid plants of the third vegetative generation produced by the method in accordance with the preferred embodiment of the present invention.

FIG. 4 illustrates morphological and floral characteristics of in vitro raised diploid plants (left) and tetraploid plants (right) according to an embodiment herein.

FIG.5 illustrates improved capitulum characteristic in tetraploid plants according to an embodiment herein.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description.

Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein. As mentioned, there is a need to satisfy the demand of edible oil consumption for overgrowing population and introduce method for producing high yielding hybrid varieties of edible oil seeds. The present invention achieves this by introducing a process for producing edible oil hybrid seeds. The method produces tetraploid plant through chemical mutagenenis. The produced tetraploid plants and their floral behavior result into more seed yield, and subsequent improvement in the productivity which also has stress tolerance ability Doubling the number of chromosomes, having more chlorophyll content and larger-sized capitulum and sepals are some of the features of the tetraploid plant resulting into high yield.
The present disclosure relates to a pure line diploid plant or shoots thereof produced from diploid seeds of Guizotia abyssinica under in vitro condition.
The present disclosure further provides a method of producing a tetraploid plant.
Referring now to the drawings, and more particularly to FIGS. 1 to 5, where similar

reference characters denote corresponding features consistently throughout the

figures, preferred embodiments are shown.

FIG. 1 a flow diagram 100 illustrates a method for producing tetraploid plants according to an embodiment herein. In an embodiment of the present disclosure, during the method of producing a tetraploid plant, at step 101, pure line of diploid plant of a plant is grown wherein the plant can be but not limited to Guizotia abyssinica Cass. The Guizotia abyssinica cass is an important edible oil seed crop which belongs to family Asteraceae. At step 102 anti-mitotic agent is applied to in vitro raised shoots of the to the diploid plant. At Step 103 the diploid treated shoot is washed with sterile distilled water for 10 mins. At step 104 the treated diploid shoot is inoculated on nutrient medium. At step 105 observing and selecting in vitro raised tetraploid shoots; and inducing roots in the in vitro raised selected tetraploid shoots is done. At step 106 chromosome number in roots and tetraploidy level in leaf by flow cytometric method of the selected tetraploid plantlets is assessed. At step 107 the selected tetraploid plant is proliferated and multiplied. At step 108 chromosome stability is assessed for future generation in the tetraploid plants produced.

In one embodiment, the plant is Guizotia abyssinica Cass wherein said diploid Guizotia abyssinica plant has a chromosome number of 2n = 30. In one embodiment, the anti-mitotic agent applied invitro to the diploid plants is colchicine. The concentration of colchicine for the treatment to the shoots of the diploid plants for induction of tetraploidy is decided according to LD50 value. The results revealed that LD50 value of colchicines concentration was appeared at 0.02%. The said colchicine solution is prepared by preparing stock solution of colchicine (1%) by dissolving 1 g of colchicine powder in 100 ml of sterile Murashige and Skoogs (MS) nutrient medium and diluting it to obtain 0.02% colchicine solution.

In another embodiment, the nutrient medium in the process of inoculation is Murashige and Skoogs (MS) medium incorporated with 6-benzylaminopurine (BAP) at concentration of 1 mg l-1.The shoots survived at LD50 value were elongated by about 2 to 3 cm within three weeks of culture on solid Murashige and Skoogs (MS) medium containing 1 mg l-1 6-benzylaminopurine (BAP).

In another embodiment, the effective method of producing tetraploids consists of treating the apical portion of in vitro raised shoots of diploid plant with liquid Murashige and Skoogs (MS) medium containing 1 mg l-1 6-benzylaminopurine (BAP) + 0.02% concentration of colchicine for 0, 4, 8, 12, and 16 hours. In yet another embodiment, the shoot proliferation nutrient medium comprises Murashige and Skoog’s salts, vitamins, 6-benzylaminopurine (BAP), indole-3-acetic acid (IAA), 3% sucrose and 0.8% agar-agar wherein the concentration of 6-benzylaminopurine (BAP) and indole-3-acetic acid (IAA) is 1 mg l-1 and 0.5 mg l-1 respectively.

In another embodiment the tetraploid plant has increased ploidy status wherein the

chromosomes in the tetraploid plant doubled, 2n = 60 due to treating apical meristem of diploid plant with liquid MS at 0.02% concentration colchicine. The effect of colchicine on generation of tetraploid plants is shown below in Table 1.
Table 1:
Colchicine treatment (%) Duration of treatment(h) Survival rate (%) No. of shoots with ploidy
2n = 2x 4x

0.0 4 100 21 -
8 100 21 -
12 100 21 -
16 100 21 -

0.02 4 47.6 7 3
8 61.9 8 5
12 52.3 6 5
16 47.6 6 4

In an embodiment, about 42% of plantlets are tetraploids at 0.02% concentration of colchicine.

In yet another embodiment the tetraploid plants of Guizotia abyssinica is confirmed by measuring the number of chromosomes which is doubled as compared to diploid
plants. Other features such as having more chlorophyll content (darker green color leaves), floral characteristics such as having large-sized sepals, increased diameter of capitulum, and reduced number of capitulum per plant also confirm the tetraploidy status of the plant.

In an embodiment, the assessment of stability of ploidy level in leaf and the chromosome number in roots of tetraploid and diploid plantlets is carried out at every subculture by the flow cytometric and chromosome count method.

The graphical representation showing leaf cell nuclear DNA content according to an embodiment herein is illustrated in FIG 2. In one embodiment, the flow cytometric histogram of diploid plant is depicted in FIG.2 (A) and the flow cytometric histogram of tetraploid plant is depicted in FIG.2 (B) wherein the histogram of tetraploid plant represents doubling the nuclear DNA content.
The flow cytometric analysis results showing DNA size and 2C DNA content in leaf tissues of diploid and novel tetraploids is shown in table 2 below.
Table 2:
Plant Sample X-Mean (Sample G1 peak mean) 2C DNA (Sample)
(pg) DNA size (Mbp)
Diploid plant 22770 4.70 4536
Tetraploid plant 42337 10.34 9974

In one embodiment, the flow histogram confirms the doubing of the DNA content of the tetraploid plant thus confirming the tetraploial status of the plant.

In another embodiment, chromosome number in leaf derived roots of tetraploid plants has doubled chromosomes as compared to leaf derived roots of diploid plants The graphical representation of number of chromosomes in leaf of derived roots of diploid and tetraploid plants of the third vegetative generation produced by the method in accordance with an embodiment of the present disclosure is illustrated in FIG 3.
Table 3 below is showing number of chromosomes per nuclei in said diploid and novel tetraploids.
Table 3:
Plant sample Number of chromosomes per nuclei
Diploid plant 2n = 2x = 30
Tetraploid plant 4x = 60

From the above, it is clear that chromosome number in leaf derived roots of tetraploid plants has doubled chromosomes as compared to leaf derived roots of diploid plants.
FIG. 4 illustrates morphological and floral characteristics of in vitro raised diploid plants and tetraploid plants according to an embodiment herein. The tetraploid plant
has increased ploidy status wherein the chromosomes in the tetraploid plant doubled

due to treating apical meristem of the diploid plant with liquid MS at 0.02% concentration colchicine.

In one embodiment the tetraploid plant of the Guizotia abyssunica Cass has doubled chromosomes, more chlorophyll content, large-sized sepals, increased diameter of
capitulum, and reduced number of capitulum per plant. The increase in the floral

characteristic also confirms the ploidy status of the Guizotia abyssunica Cass.

FIG.5 illustrates improved capitulum characteristic in tetraploid plants according to an embodiment herein. The tetraploid plant of Guizotia abyssunica Cass has improved capitulum as shown in the figure. A head or the capitulum is a short
dense spike in which the flowers are borne directly on a broad, flat peduncle, giving inflorescence the appearance of a single flower.

In one embodiment the tetraploid plants of Guizotia abyssinica is confirmed by measuring the number of chromosomes which is doubled as compared to the diploid plants with more chlorophyll content (darker green color), and in floral characteristics such as having more large-sized sepals, increased diameter of
capitulum, and reduced number of capitulum per plant. Said characteristics also confirm the tetraploidy status of the Guizotia abyssinica Cass. The produced tetraploid plants and their floral behavior result in more seed yield, and subsequent improvement in the productivity and stress tolerance ability.
EXAMPLES:

EXAMPLE 1: Producing tetraploid plants of Guizotia abyssinica Cass:

1.1 Materials Preparation:
In the present method of producing tetraploid plants of Guizotia abyssinica Cass, the certified seed material of Guizotia abyssinica Cass cv. Sahyadri (IGP-76) was procured from Mahatma Phule Agricultural University’s, Zonal Agricultural Research Station, Western region, Igatpuri, Nashik, Maharastra, India. Throughout the investigation, Murashige and Skoogs (MS) medium was used for establishment and maintenance of in vitro cultures of Guizotia abyssinica. Anti-mitotic agent, Colchicine and 6-benzyladenine (BAP) and indole-3-acetic acid (IAA) were obtained from Hi-media (Mumbai, India) and used in the experiment. Stock solution of colchicines (1%) was prepared by dissolving 1 g of colchicines powder in 100 ml of sterile MS nutrient medium. Then from stock solution, 0.005, 0.01, 0.02, 0.03, and 0.04% concentration solution of colchicines were prepared on dilutions using MS nutrient medium.
1.2 Process of producing tetraploid plants of Guizotia abyssinica Cass
In the present method of producing tetraploid plants of Guizotia abyssinica Cass, at step i) of the method of the present disclosure, a pure line diploid plant, Guizotia abyssinica, is grown in vitro. At step ii) of the method of the present disclosure an anti-mitotic agent, 0.02% colchicine solution was applied to apical portion of in vitro raised shoots of the diploid plant. At step iii) of the method of the present disclosure, diploid plant of said plant was treated with sterile distilled water for 10 minutes; At step iv) of the method of the present disclosure said treated diploid plant was inoculated on nutrient medium. The apical meristem was treated by inverting the shoots in test tubes containing liquid Murashige and Skoogs (MS) medium with 1 mg l-1 6-benzylaminopurine (BAP) and 0.02 concentration of colchicines for 0, 4, 8, 12, and 16 hours (refer fig 1 A). The treatments were arranged in 3 replicates with 21 explants per replication. The MS medium with 1 mg l-1 BAP but not containing colchicines served as a control. The survival percentage was dependent on colchicine concentrations and duration of treatments (refer table 1 above). The shoots survived at LD50 value were elongated by about 2 to 3 cm within three weeks of culture on solid Murashige and Skoogs (MS) medium containing 1 mg l-1 6-benzylaminopurine (BAP).
At step v) of the method of the present disclosure, observing and selecting in vitro raised tetraploid shoots; inducing roots in the in vitro raised selected tetraploid shoots was carried out. At step vi) of the method of the present disclosure, assessing the chromosome number in roots and tetraploidy level in leaf by flow cytometric method of the selected tetraploid plantlets was carried out. At step vii) of the method of the present disclosure, proliferating and multiplying said selected tetraploid plants was carried out. Said shoot proliferation nutrient medium comprises Murashige and Skoog’s salts, vitamins, 6-benzylaminopurine (BAP), indole-3-acetic acid (IAA), 3% sucrose and 0.8% agar-agar wherein the concentration of 6-benzylaminopurine (BAP) and indole-3-acetic acid (IAA) is 1 mg l-1 and 0.5 mg l-1 respectively. At step viii) of the method of the present disclosure, assessing the chromosome stability in future generations of said tetraploid plants produced was carried out.

The various steps involved in producing tetraploid plants of Guizotia abyssinica Cass in accordance with an embodiment of the present disclosure is represented as a flowchart in Figure 1. The produced tetraploid plant has increased ploidy status wherein the chromosomes in the tetraploid plant has doubled, has more chlorophyll content, large-sized sepals, increased diameter of capitulum, and reduced number of capitulum per plant. (Refer FIG 3, 4 and 5).

EXAMPLE 2: Analysis of Producing tetraploid plants of Guizotia abyssinica Cass:

2.1: Analysis of Nuclear DNA content:

Nuclear DNA content was determined using flow cytometric analysis for confirmation of increased ploidy level in leaf samples of four months old in vitro raised colchicine treated shoots (refer FIG 2). In brief the procedure was following as: The leaf samples (0.5 g) were crushed in ice-cold Galbraith’s buffer solution together with 0.1 % v/v Triton-X100. The homogenate was filtered through nylon syringe micro-filter. The filtrate was added in tube containing RNAse A (2.5 µl) and incubated at 4°C for 10 min. The nuclear DNA was stained by adding 50 µl/ml of PI and the suspensions were incubated in dark for 30 min prior to analysis. The Attune® Acoustic Focusing Flow Cytometer (Life Technologies, Thermo Fisher Scientific) was used to measure the fluorescence intensity of samples. The nuclear genome size was determined using following formula: 2C DNA content (pg) = sample G1 peak mean x standard 2C DNA content (pg) / standard G1 peak mean.

2.2: Analysis of chromosome number:
The tetraploid nature of plantlets was also confirmed by counting the chromosome number in root cells. Aceto-carmine stain was used for chromosome staining. The method is in brief: Aceto-carmine powder was added to the boiling acetic acid (45%) and the mixture was boiled for 5 min with occasional stirring until colour becomes dark red. The suspension was allowed to cool, filtered through whatman filter paper and stored in an amber color bottle, at 4°C until use. Para-dichlorobenzene (PDB) (3 g) dissolved in distilled water (200ml) and incubated overnight at 60°C and gently stirred before use. At early morning (6.00 to 8.00 am), the root tips of diploid and colchicines treated plantlets were fixed for 4 hr in PDB solution at 4°C. Before use, the root tips were washed in distilled water and then hydrolyzed in 1.0 N HCl at 60°C for 2 min and rinsed with distilled water.

For squash preparation and chromosome staining, the hydrolysed root tip (5 to 7 mm) was placed on a glass slide in a drop of 1% aceto-carmine stain for 2 min. A cover glass was placed over the stained root tips and thumb pressure was applied slowly to spread the cells especially dividing the cells. The cells in squash were observed for chromosome number under light microscope (Leica Microsystems DM 3000 LED, Germany) using 40 x and 100 x magnifications.

The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein has been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the application.
,CLAIMS:We Claim:

1. A pure line diploid plant or shoots thereof produced from diploid seeds of Guizotia abyssinica under in vitro condition.
2. The plant or plant part as claimed in claim 1, wherein said diploid Guizotia abyssinica plant has a chromosome number of 2n = 30.
3. A method of producing a tetraploid plant, said method comprising the steps of:
i) growing a pure line diploid plant in vitro of said plant, wherein the plant is an edible oil seed crop belonging to family Asteraceae but not limited to Guizotia abyssinica;
ii) applying an anti-mitotic agent to apical portion of in vitro raised shoots of the diploid plant;
iii) treating the diploid plant of said plant with sterile distilled water for 10 minutes;
iv) inoculating said treated diploid plant on nutrient medium;
v) observing and selecting in vitro raised tetraploid shoots; inducing roots in the in vitro raised selected tetraploid shoots;
vi) assessing the chromosome number in roots and tetraploidy level in leaf by flow cytometric method of the selected tetraploid plantlets;
vii) proliferating and multiplying said selected tetraploid plant; and
viii ) assessing the chromosome stability in future generations of said tetraploid plants produced.
4. The method as claimed in claim 3, wherein said anti-mitotic agent is colchicine; wherein the concentration of colchicine for the treatment to the shoots of the diploid plants for induction of tetraploidy is decided according to LD50 value and wherein said effective concentration of anti-mitotic agent for generation of tetraploid plant is low concentration of colchicine.
5. The method as claimed in any one of claims 3 or 4, wherein said effective low concentration of anti-mitotic agent for generation of tetraploid plant is 0.02% and wherein the said solution is prepared by preparing stock solution of colchicine (1%) by dissolving 1 g of colchicine powder in 100 ml of sterile Murashige and Skoogs (MS) nutrient medium and diluting it to obtain 0.02% colchicine solution.
6. The method as claimed in any one of claims 3 to 6, wherein about 42% of plantlets are tetraploids at 0.02% concentration of colchicine.
7. The method as claimed in claim 3, wherein said nutrient medium in the process of inoculation is Murashige and Skoogs (MS) medium incorporated with 6-benzylaminopurine (BAP) at concentration of 1 mg l-1.

8. The method as claimed in claim 3, wherein said effective method of producing tetraploids consists of treating the apical portion of in vitro raised shoots of diploid plant with liquid Murashige and Skoogs (MS) medium containing 1 mg l-1 6-benzylaminopurine (BAP) + 0.02% concentration of colchicine for 0, 4, 8, 12, and 16 hours.

9. The method as claimed in claim 3, wherein said shoot proliferation nutrient medium comprises Murashige and Skoog’s salts, vitamins, 6-benzylaminopurine (BAP), indole-3-acetic acid (IAA), 3% sucrose and 0.8% agar-agar wherein the concentration of 6-benzylaminopurine (BAP) and indole-3-acetic acid (IAA) is 1 mg l-1 and 0.5 mg l-1 respectively.

10. The method as claimed in claim 3, wherein said tetraploid plant has increased ploidy status; and wherein the chromosomes is doubled in said tetraploid plant 2n= 60.

11. The method as claimed in claim 3, wherein the assessment of stability of ploidy level in leaf and the chromosome number in roots of tetraploid and diploid plantlets is carried out at every subculture by the flow cytometric and chromosome count method.

12. A tetraploid plant produced by the method as defined in claim 3.
13. The plant as claimed in claim 13, wherein said tetraploid plant has increased ploidy status (doubled chromosomes); increase chlorophyll content (darker green color leaves), and in floral characteristics such as having more large-sized sepals, increased diameter of capitulum, and reduced number of capitulum per plant resulting in more seed yield, and subsequent improvement in the productivity and stress tolerance ability.