WO2010007628A1

WO2010007628A1 - Method for enhancing regeneration frequency of brassica species

Info

Publication number: WO2010007628A1
Application number: PCT/IN2009/000402
Authority: WO
Inventors: Ashwani Pareek; Gautam Kumar Roy
Original assignee: Ashwani Pareek; Gautam Kumar Roy
Priority date: 2008-07-15
Filing date: 2009-07-14
Publication date: 2010-01-21

Abstract

The present invention relates to a method for enhancing the regeneration frequency of varieties of Brassica sps., wherein the method is characterized by comprising treatment of hypocotyls explants segments of Brassica sps. with a medium comprising Benzyl Amino Purine and Napthalene Acetic Acid [B₁N₁ medium], wherein the treatment results in fast callus initiation, root hairs initiation, appearance of green spots in the callus so formed, and granulation of callus so formed, and appearance of anthocyanin pigmentation in few species of Brassica.

Description

METHOD FOR ENHANCING REGENERATION FREQUENCY OF

BRASSICA SPECIES

Field of the Invention:- The present invention relates to the field of method and medium for improving crop by improving regeneration frequency of the Brassica sps. Particularly, it relates to the field of method and medium for improving economical value of the Brassica sps. by improving its regeneration frequency. In particular, the present invention relates to a method for enhancing regeneration frequency of the Brassica sps. It also relates to the regeneration medium capable of being used in method for improving regeneration frequency of the Brassica sps. Background of the Invention:-

The Brassica oil seed crop belongs to the same taxonomic family as Arabidopsis thaliana. Economically, Brassica is loosely categorized as oilseed, vegetable, and condiment crop. The Brassica napus, Brassica juncea, and Brassica carinata provide about 12-15% of the worldwide edible vegetable oil supplies. The oil from Brassica nigra, commonly known as "rai" or "black mustard" is used for routine cooking and also as cough suppressant and to cure respiratory diseases. The rapeseed varieties are suitable for farmers of the coastal wetland that is capable of playing important role in oil and vegetable productivity. The Brassica species are a valuable source of dietary fiber, vitamin C, and other possible salubrious factors such as anticancer compounds.

In-addition to above, there are multipurpose uses of Brassica sps., for example Mustard Oil, Green vegetables, condiment etc. Accordingly, several attempts have been made to enhance regeneration frequency of the Brassica sps. in both cultivated and wild species. The recent reports for regeneration in B. napus have documented the highest percentage of shoot regeneration frequency of 200% obtained in 21 days old explants [Mousavi et al 2005, Biologia Plantarum 49:175-180]. The B. nigra has been attempted for regeneration from both callus and protoplast [Gupta et al 1990 Plant Cell Reports 9:427-430].

However, these attempts have been made by adopting the conventional regeneration methodology using the hypocotyls of Brassica species, which, as evident from organogenesis of variety of explants of Brassica species, have been found largely to be genotype dependent and species specific. It has been shown that regeneration potential is heritable, and hence, a genetically controlled trait (Bhojwani et at, 1984; Templeton-Sommers and Collins, 1986). Therefore, methods known in the art have been found to be variety specific.

The genetic improvement of Brassica species has been primarily achieved through conventional breeding methods. However, such efforts are restricted to species that are sexually compatible.

The adoption of new technologies, such as recombinant DNA technology and genetic engineering, which are not species specific is preferred choice. Further, with more and more gene information being made available, more and more efforts are underway for genetic improvement of cultivated Brassica species. However, the adoption of new technologies and the genetic improvement of cultivated Brassica species will be more convenient and economical, if an efficient regeneration process, which should be suitable for various species and not for one species is made available.

The identification of responding species is a critical factor for achieving success in regeneration as well as genetic transformation in Brassica sps. This is so, because the conventionally developed regeneration processes are for a particular species with a particular cultivar, which is again very specific requirement, and hence, these processes are also limited in application. Furthermore, such methods have not been found suitable, if one is interested in both - the regeneration and genetic transformation of cultivars which are tolerant to stresses such as salinity and/or drought, that is, salt sensitivity as well as salt tolerance var. of Brassica sps.

Therefore, availability of a suitable regeneration process, which is capable of enhancing regeneration frequency of varieties of Brassica sps. and not for one species of Brassica would go a long way in achieving these targets. Further, inspite of the success obtained with development of species specific regeneration media, there is no disclosure of a regeneration medium, which is suitable for enhancing regeneration frequency of varieties of Brassica sps.

Therefore, availability of a suitable regeneration medium, which is capable of enhancing regeneration frequency of varieties of Brassica sps. and not for one species of Brassica would go a long way in achieving above targets.

It is clear from the foregoing description that it would be highly desirable to have a regeneration process and the regeneration medium therefor, which are capable of enhancing regeneration frequency, and hence, capable of enhancing the economical value not only of one Brassica sps., but of more than one Brassica sps. As elaborated herein above, it is anticipated that such regeneration medium and the regeneration processes employing the same to enhance the regeneration frequency, and hence, the economical value of the Brassica sps. will aid further support to several ongoing research and development work where genetic transformation of Brassica sps. for improvement of desired trait is being attempted. Need of the Invention :-

Therefore, there is a need to have a regeneration process and a regeneration medium therefor, which are capable of enhancing regeneration frequency, and hence, are capable of enhancing the economical value of more than one Brassica sps. and not merely of one Brassica sps., which means which are not species specific, so that the genetic transformation of Brassica sps. for improvement of desired trait can also be achieved. Objects of the Invention :-

Accordingly, the main object of the present invention is to provide a regeneration process and a regeneration medium therefor, which are capable of enhancing regeneration frequency, and hence, are capable of enhancing the economical value of more than one Brassica sps. and not merely of one Brassica sps., which means which are not species specific, so that the genetic transformation of Brassica sps. for improvement of desired trait can also be achieved, and therefore, the drawbacks and limitations of the prior art as described herein above may be overcome.

Therefore, the object of the present invention is to provide a regeneration process, which is capable of enhancing regeneration frequency, and hence, the economical value not only of one Brassica sps., but of more than one Brassica sps. The another object of the present invention is to provide a regeneration medium, which is capable of enhancing regeneration frequency, and hence, the economical value not only of one Brassica sps., but of more than one Brassica sps.

This is also an object of the present invention to provide a regeneration process, being capable of enhancing regeneration frequency of more than one Brassica sps so that the genetic transformation of Brassica sps. for improvement of desired trait can also be achieved.

This is also an object of the present invention to provide a regeneration process for enhancing regeneration frequency of varieties of Brassica sps. including, but not limited to Brassica napus, Brassica juncea, Brassica carinata, and Brassica nigra in order to enhance their economical values, contrary to conventional methods, which are species specific.

This is also an object of the present invention to provide a regeneration process for enhancing regeneration frequency of varieties of Brassica sps., which is capable of producing the genetically modified plant as well as transgenic plant.

This is also an object of the present invention to provide a regeneration process for enhancing regeneration frequency of varieties of Brassica sps., wherein the method is capable of enhancing the economical value of varieties of Brassica sps. by obtaining the enhanced regeneration frequency of about 40 to about 60% as compared to conventionally known processes.

This is also an object of the present invention to provide a regeneration medium, being capable of enhancing regeneration frequency of more than one Brassica sps. so that the genetic transformation of Brassica sps. for improvement of desired trait can also be achieved. This is also an object of the present invention to provide a regeneration medium for enhancing regeneration frequency of varieties of Brassica sps. including, but not limited to Brassica napus, Brassica juncea, Brassica carinata, and Brassica nigra in order to enhance their economical values, contrary to conventional methods, which are species specific. This is also an object of the present invention to provide a regeneration medium for enhancing regeneration frequency of varieties of Brassica sps., which is capable of producing the genetically modified plant as well as transgenic plant.

This is also an object of the present invention to provide a regeneration medium for enhancing regeneration frequency of varieties of Brassica sps., wherein the medium is capable of enhancing the economical value of varieties of Brassica sps. by obtaining the enhanced regeneration frequency of about 40 to about 60% as compared to the one obtained by employing conventionally known media.

Other objects and advantages of the present invention will be more apparent when following description is read in conjunction with the accompanying figures, which are not intended to limit scope of the present invention. Brief Description of the Accompanying Figures:-

Figure 1 illustrates process steps of regeneration process in accordance with one embodiment of the present invention. Figure 2 illustrates process steps of regeneration process, wherein transformation process is combined in accordance with another embodiment of the present invention. Description and Preferred Embodiments of the Invention:- As it is clear from the foregoing description that it would be highly desirable to have a regeneration process and the regeneration medium therefor, which are capable of enhancing regeneration frequency, and hence, the economical value not only of one Brassica sps., but of more than one Brassica sps., preferably of about four Brassica sps.. Therefore, the present invention aims at providing a regeneration process and a regeneration medium therefor which are capable of enhancing the regeneration frequency of varieties of Brassica sps. The present invention also aims at providing the culture conditions for regeneration, proliferation and rooting of callus of Brassica sps. by a judicious selection of explants along with suitable combinations of hormones in the culture medium so that the above-described drawbacks and limitations of the prior art can be overcome.

With above aim, the inventors have found that if hypocotyl explants segments, obtained from hypocotyls of seedlings of Brassica sps. incubated in MSO Medium [Murashige and Skoog's medium], are treated with a medium comprising Benzyl Amino Purine and Napthalene Acetic Acid [B₁N₁ (BAP and NAA) medium], the callus initiation surprisingly starts just within about 3 to 5 days, and the root hairs initiation surprisingly starts from callus mass formation just within about 5 to 10 days, and the green spots in the callus surprisingly start appearing after another about 12 to 20 days, and within next about 12 to 20 days the callus so formed surprisingly starts appearing granular, and even anthocyanin pigmentation is also simultaneously observed in few species of Brassica. The inventors have also found that if green portion of the callus so formed is transferred to fresh batch of B₁N₁ medium, it surprisingly results in very fast growth and just within about 10 days from such transfer, these green spots develop into small plantlets, which upon treatment with a medium comprising Murashige and Skoog's medium and Indole Acetic Acid [MI (MS + IAA) medium] surprisingly results in enhancement of root development resulting in development of plants with developed root system, which have been found to be capable of growing as hardened plants in the green house. The inventors have also found that this process to produce the transgenic plant is surprisingly universal, and hence, applicable to more than one Brassica Sps. Furthermore, about 40% to about 60% enhancement has been observed in the regeneration frequency of Brassica Sps.

The inventors have also found that if hypocotyl explant segments, obtained from hypocotyls of seedlings of Brassica sps. and incubated with a medium comprising Benzyl Amino Purine and Napthalene Acetic Acid [B₁N₁ (BAP and NAA) medium] for check of any infection are treated [infected] with Agrobacterium culture containing the gene of interest on liquid B₁N₁ medium, the gene of interest surprisingly gets transferred to Brassica Sps. and the plant thus produced has been found to be genetically modified plant. Furthermore, about 40% to about 60% enhancement has also been observed in the regeneration frequency of Brassica sps. while producing genetically modified plant.

Accordingly, the present invention relates to a method for enhancing the regeneration frequency of varieties of Brassica sps., wherein the method is characterized by comprising treatment of hypocotyls explants segments of Brassica sps. with a medium comprising Benzyl Amino Purine and Napthalene Acetic Acid [B₁Ni medium], wherein the treatment results in fast callus initiation, root hairs initiation, appearance of green spots in the callus so formed, and granulation of callus so formed, and appearance of anthocyanin pigmentation in few species of Brassica. In accordance with preferred embodiment of present invention, the method further comprises treating green portion of the callus after granulation of callus and appearance of anthocyanin pigmentation in few species of Brassica with fresh batch of a medium comprising Benzyl Amino Purine and Napthalene Acetic Acid [fresh batch OfBiN₁ medium], wherein the treatment results in fast growth of plantlets. In accordance with another preferred embodiment of present invention, the method further comprises treating plantlets of Brassica Sps. with a medium comprising Murashige and Skoog's medium and Indole Acetic Acid [MI (MS + IAA) medium], wherein the treatment results in enhancement of root development resulting in developed plantlets having developed root system, which are capable of growing as hardened plants in the green house as the transgenic plant.

In accordance with one of the preferred embodiments of the present invention, it relates to a method for enhancing the regeneration frequency of varieties of Brassica sps., wherein the method comprises treating hypocotyls explants segments of Brassica sps. with a medium comprising Benzyl Amino Purine and Napthalene Acetic Acid [B₁Ni medium] to result in fast callus initiation, root hairs initiation, appearance of green spots in the callus so formed, and granulation of callus so formed, and appearance of anthocyanin pigmentation in few species of Brassica, followed by treating green portion of the callus after granulation of callus and appearance of anthocyanin pigmentation in few species of Brassica with fresh batch of a medium comprising Benzyl Amino Purine and Napthalene Acetic Acid [fresh batch of B₁N₁ medium] to result in fast growth of plantlets, followed by treating plantlets of Brassica Sps. with a medium comprising Murashige and Skoog's medium and Indole Acetic Acid [MI (MS + IAA) medium] to result in enhancement of root development resulting in developed plantlets having developed root system, which are capable of growing as hardened plants in the green house as the transgenic plant.

In accordance with one of the preferred embodiments of present invention, the method further comprises incubating hypocotyls explants segments with a medium comprising Benzyl Amino Purine and Napthalene Acetic Acid [B₁N₁ medium] for check of any infection, and thereafter treating [infecting] with Agrobacterium culture containing the gene of interest on liquid B₁N₁ medium before the treatment of hypocotyls explants segments with B₁N₁ medium, wherein the treatment results in transfer of gene of interest to Brassica sps, and the plant produced is the genetically modified plant. Accordingly, in one embodiment, the present invention relates to a method for enhancing the regeneration frequency of varieties of Brassica sps., wherein the method is characterized by comprises incubating hypocotyls explants segments, obtained from hypocotyls of seedlings of Brassica sps. , with a medium comprising Benzyl Amino Purine and Napthalene Acetic Acid [B₁N₁ medium] for check of any infection, and thereafter, treating [infecting] with Agrobacterium culture containing the gene of interest on liquid BiN₁ medium, wherein the treatment results in transfer of gene of interest to Brassica sps, followed by treating Agrobacterium treated hypocotyls with a medium comprising Benzyl Amino Purine and Napthalene Acetic Acid [B₁N₁ medium] to result in fast callus initiation, root hairs initiation, appearance of green spots in the callus so formed, and granulation of callus so formed, and appearance of anthocyanin pigmentation in few species of Brassica, followed by treating green portion of the callus after granulation of callus and appearance of anthocyanin pigmentation in few species of Brassica with fresh batch of a medium comprising Benzyl Amino Purine and Napthalene Acetic Acid [fresh batch of BjNi medium] to result in fast growth of plantlets, followed by treating plantlets of Brassica Sps. with a medium comprising Murashige and Skoog's medium and Indole Acetic Acid [MI (MS + IAA) medium] to result in enhancement of root development resulting in developed plantlets having developed root system, which are capable of growing as hardened plants in the green house, and wherein the plant produced is the genetically modified plant.

In accordance with one of the preferred embodiments of the present invention, the method further comprises steps of:- a). transferring the plants having the developed root system to soil; b). covering the plants of step — a) for hardening; and c). transferring the hardened plants from step — b) to the green house.

In accordance with one of the preferred embodiments of the present invention, the hypocotyls explants segments are obtained from the hypocotyls of the seedlings incubated in Murashige and Skoog's medium [MSO Medium]. In accordance with one of the preferred embodiments of the present invention, the MSO medium comprises gamborg vitamin, which has been found to enhance germination of seeds.

In accordance with one of the embodiments of the present invention, the Benzyl Amino Purine is 6-Benzyl Amino Purine. In accordance with one of the preferred embodiments of the present invention, the callus initiation and root hairs initiation start within a period varying upto about 15 days from the day of start of treatment. In accordance with one of the preferred embodiments of the present invention, the callus initiation starts within a period varying from about 3 to about 5 days from the day of start of treatment, and root hairs initiation starts within a period varying from about 5 to about 10 days from the callus mass so formed.

In accordance with one of the preferred embodiments of present invention, the appearance of green spots in the callus mass having root hairs starts within a period varying from about 12 to about 20 days after the root hairs initiation in the callus, and callus starts appearing granular in another period varying from about 12 to about 20 days after appearance of green spots, and formation of anthocyanin pigmentation in few species of Brassica Sps. starts simultaneously to granulation of callus mass.

In accordance with one of the preferred embodiments of present invention, the treatment of green portion [spots] of the callus with fresh batch Of B₁N₁ medium has been found to result in fast development of small plantlets within a period varying upto about 10 days from start of such treatment.

In accordance with one of the preferred embodiments of present invention, the treatment of hypocotyls explants segments of Brassica sps. with B₁N₁ medium is carried out under light in a manner that each container contains about 35 to about 45 explants and the container is sealed and kept at about 20 to 25°C, which has been found to result in fast callus initiation, root hairs initiation, appearance of green spots in the callus so formed, and granulation of callus so formed, and appearance of anthocyanin pigmentation in few species of Brassica. In accordance with one of the preferred embodiments of present invention, the light for treatment of hypocotyls explants segments of Brassica sps. with B₁N₁ medium is cool white fluorescent light, which has been found to result in comparatively faster induction of callus and initiation of root hairs.

In accordance with one of the preferred embodiments of present invention, the hypocotyls are cut into segments having a length of about 5 to 7 mm, which has been found to result in good contact with the medium.

In accordance with one of the preferred embodiments of the present invention, the hypocotyls being treated with Agrobacterium culture are simultaneously treated with antibiotic solution-augmentin. In accordance with one of the preferred embodiments of the present invention, the treatment with fresh lot of BjN₁ medium is carried out in presence of antibiotic solution.

In one embodiment, the present invention also relates to regeneration medium which is capable of enhancing regeneration frequency of varieties of Brassica sps. meaning thereby for enhancing economical value of varieties of Brassica sps.

In accordance with present invention, the seedlings of Brassica sps. and preparation of their hypocotyls explants segments may be grown in any conventional manner. However, in accordance with one of the preferred embodiments of the present invention, the seedlings of Brassica sps. and their hypocotyls explants segments are grown by a process comprising following steps:- a) washing the Brassica sps. seeds with water followed by treatment with HgCl₂ solution, and then with ethanol, wherein HgCl₂ solution is taken in about 0.05% HgCl₂ concentration and ethanol taken is about 70% ethanol; b) rinsing the washed seeds from step - a) to remove the traces of HgCl₂ and ethanol; c) drying the rinsed seeds from step - b) prior to inoculation in the MSO medium, wherein the MSO medium has pH of about 5.8; d) germinating the dried seeds from step - c) by incubation for development of seedlings, wherein preferably about 10 seeds are germinated per culture tube to avoid crowding, wherein the germination is carried out by incubating the seeds at a temperature of about 20⁰C to 25⁰C under dark condition for about 2 days followed by placing the culture tubes under light conditions; e) taking the hypocotyls of seedlings from step - d) for callus induction and regeneration, wherein the hypocotyls preferably of about 5 to 6 days old seedlings are taken, wherein the hypocotyls taken have a length varying from about 4 to 5 cm; f) removing the seedlings of step — e) from the culture tubes under sterile conditions, and cutting the hypocotyls into hypocotyls explants segments for treatment or incubation with B₁N₁ (BAP and NAA) medium, wherein the hypocotyls are cut into hypocotyls explants segments of a length varying from about 5 to about 7 mm.

In accordance with one of the 'preferred embodiments of the present invention, Benzyl Amino Purine and Napthalene Acetic Acid medium [B₁N₁ (BAP and NAA) medium], the fresh batch Of B₁N₁ medium and liquid B₁N₁ medium as referred herein comprise Benzyl Amino Purine and Napthalene Acetic Acid, wherein each are taken in an equal amount, preferably in an amount of about 2mg per L.

In accordance with one of the preferred embodiments of the present invention, the Murashige and Skoog's medium and Indole Acetic Acid [MI (MS + IAA)] medium, wherein each of Murashige and Skoog's medium and Indole Acetic Acid are taken in equal amount, preferably in an amount of about 2mg per L.

Accordingly, in accordance with one of the preferred embodiments of the present invention, the regeneration process to have transgenic plant comprises the steps of:-

1. treating the hypocotyls explants segments with B₁N₁ (BAP and NAA) medium and keeping under light for the induction of callus and initiation of root hairs, which is found to results in fast initiation of callus and root hairs; 2. start of appearance of green spots in the callus of step - 1, wherein the callus starts appearing granular and often anthocyanin pigmentation is observed in few species of Brassica;

3. transferring the green portion of the callus of step - 2 to fresh batch of B₁N₁ medium, which is found to results in very fast growth and these green spots develop into small plantlets;

4. sub-culturing the plantlets from step - 3 on MI (MS + IAA) medium to enhance the root development, which is found to result in fast root development, and thereby of the plants having the developed root system. The present process is now described with the help of accompanying Figure 1, for the better illustrations and understanding and it is not intended to limit scope of the present invention.

In accordance with one of the preferred embodiments of the present invention, the regeneration process to have transgenic plant [Fig. 1] comprises the following steps :-

1. talcing the hypocotyls of about 5 to 6 days old seedlings for callus induction and regeneration (Fig. Ia.);

2. removing the seedlings with hypocotyls of step - 2 from the culture tubes, and cutting the hypocotyls into hypocotyls explants segments; 3. transferring the hypocotyl explants from step — 3 to B₁N₁ (BAP and NAA) medium in a manner that it touches the medium and keeping under light for the induction of callus and initiation of the root hairs. It is observed that callus initiation starts within about 3 to 5 days (Fig. Ib.), and the root hairs initiation starts from callus mass within about 5 to 10 days (Fig. Ic); 4. start of appearance of green spots in the callus of step - 3. It is observed that after another about 12 to 20 days the green spots starts appearing in the callus, and within next about 12 to 20 days, the callus starts appearing granular and often anthocyanin pigmentation is observed in few species of Brassica;

5. transferring the green portion of the callus of step - 4 after another about 1 month period to fresh batch of B₁N₁ medium, which surprisingly results in very fast growth and within about 10 days these green spots develop into small plantlets (Fig. Id.);

6. sub-culturing the plantlets from step - 5 on a MI (MS + IAA) medium to enhance the root development (Fig.le-f.); 7. transferring the plants from step — 6, particularly when the plants show developed root system to the soil after washing the roots thereof;

8. covering the plants of step - 7 for hardening (Fig.1 g.); and

9. transferring the hardened plants from step - 8 to the green house after a period of about 12 to about 15 days.

In accordance with one of the particular embodiments of the present invention, the regeneration process to have transgenic plant [Fig. 1] comprises the following steps :-

1. taking the hypocotyls having a length varying from about 4 to about 5 cm of about 5 to 6 days old seedlings for callus induction and regeneration (Fig. Ia.);

2. removing the seedlings of step - 1 from the culture tubes under sterile conditions, and cutting the hypocotyls into segments having a length of about 5 to 7 mm (hereinafter referred to as explants);

3. transferring the hypocotyls explants from step - 2 to a container containing B₁N₁ (BAP and NAA) medium at about 40 to about 45 explants per container, which is sealed and kept at about 20 to 25°C under the light for the induction of callus and initiation of root hairs. It is observed that callus initiation starts within about 3 to 4 days (Fig. Ib.), and the root hairs initiation starts from callus mass within about 5 to 10 days (Fig. Ic); 4. start of appearance of green spots in the callus of step - 3. It is observed that after another about 12 to 20 days the green spots start appearing in the callus, and within next about 12 to 20 days, the callus starts appearing granular and often anthocyanin pigmentation is observed in few species of Brassica;

5. transferring the green portion of the callus of step - 4 after another about 1 month period to fresh batch of B₁N₁ medium, which surprisingly results in very fast growth of green spots and within about 10 days these green spots develop into small plantlets (Fig. Id.);

6. sub-culturing the plantlets from step - 5 in a container on a MI (MS + IAA) medium to enhance the root development (Fig.le-f.); 7. transferring the plants from step - 6, particularly when the plants show developed root system to the soil after washing the roots thereof; 8. covering the developed plantlets having enhanced roots from step - 7 for hardening (Fig. Ig.); and 9. transferring the hardened plants from step — 8 to the green house after a period of about 12 to about 15 days.

In one embodiment of the present invention, it relates to production of genetically modified plant. The genetically modified plant is produced when above described process is combined with transformation, for example, if regeneration process comprises following steps:-

a -» b' -» c' -»d' -» e,

then the process combining the feature of transformation comprises following steps:-

a -> (Bl^ B2^B3^ B4}^b' ^■» c' -»d' -» e,

wherein process steps {Bl -¥ B2-> B3-^B4} are carried out as per second embodiment of the present invention for transforming the callus by Agrobacterium mediated transformation.

It may be noted that any of the strains of Agrobacterium twnefaciens capable of transferring genetic material to Brassica species can be used in combination with the above described process or its variations. However, in accordance with one of the preferred embodiments of the present invention, particularly the improved transformation, recovery, and regeneration can be achieved by using A. tumefaciens strain GV3101 available commercially. ["The promoter of the T_L-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector". MoI Gen. Genet. 1986, 204, 383-396].

Accordingly, in second embodiment of the present invention, an efficient, stable and reproducible regeneration method is disclosed which is capable of improving economical values of the Brassica sps. by enhancing regeneration frequency thereof, and is also capable of genetically modifying the plant. In accordance with one of the embodiments of the present invention, the embodiment comprising combination of regeneration and transformation process to have genetically modified plant [Fig. 1 and Fig. 2] comprises the following steps:- 1. taking the hypocotyls having a length varying from about 4 to about 5 cm of about 5 to 6 days old seedlings for callus induction and regeneration (Fig. Ia.); 2. removing the seedlings of step - 1 from the culture tubes under sterile conditions, and cutting the hypocotyls into segments having a length of about 5 to 7 mm (hereinafter referred to as explants);

3. transferring the hypocotyls explants from step - 2 to B₁N₁ (BAP and NAA) medium and incubating to check for any infection under shaking conditions

(Fig. 2Bl).

4. co-infecting the chopped hypocotyls from step — 3 with Agrobacterium culture containing the gene of interest on liquid B₁N₁ (BAP and NAA) medium (Fig. 2B2); 5. Co-cultivating the hypocotyls from step - 4 with antibiotic solution-augmentin to remove extra Agrobacterium followed by washing (Fig. 2B3,4);

6. transferring the washed hypocotyls from step - 5 to a fresh batch of B₁N₁ (BAP and NAA) medium containing antibiotic selection for the induction of callus and initiation of root hairs (Fig. 2b'). It is observed that callus initiates within about 3 to 4 days and root hairs initiate from callus mass within about 5 to 10 days (Fig. 2c');

7. start of appearance of green spots in the callus of step — 6. It is observed that after another about 12 to 20 days the green spots start appearing in the callus, and within next about 12 to 20 days, the callus starts appearing granular and often anthocyanin pigmentation is observed in few species of Br assica;

8. transferring the green portion of the callus of step - 7 to fresh batch of B₁N₁ medium, which surprisingly results in very fast growth of green spots and within about 10 days these green spots develop into small plantlets (Fig. 2d');

9. sub-culturing the plantlets from step - 8 on a MI (MS + IAA) medium to enhance the root development (Fig.1 e-f.);

10. transferring the plants from step - 9 to the soil, particularly when the plants show developed root system;

11. covering the developed plantlets having enhanced roots from step - 10 for hardening (Fig. Ig.); and 12. transferring the hardened plants from step - 11 to the green house after a period varying from about 12 to about 15 days.

In accordance with one of the particular embodiments of the present invention, the embodiment comprising combination of regeneration and transformation process to have genetically modified plant [Fig. 1 and Fig. 2] comprises the following steps:- 1. talcing the hypocotyls having a length varying from about 4 to about 5 cm of about 5 to 6 days old seedlings for callus induction and regeneration (Fig. Ia.);

2. removing the seedlings from step - 1 from the culture tubes under sterile conditions, and cutting the hypocotyls into segments having a length of about 5 to 7 mm (hereinafter referred to as explants);

3. transferring the hypocotyl explants from step - 2 to a container containing B₁N₁ (BAP and NAA) medium at about 40 to about 45 explants per container and are incubated overnight to check for any infection under shaking conditions (Fig. 2Bl). 4. co-infecting the chopped hypocotyls from step - 3 with Agrobacterium culture containing the gene of interest on liquid B₁N₁ (BAP and NAA) medium the next day (Fig. 2B2);

5. Co-cultivating the hypocotyls from step - 4 with antibiotic solution-augmentin to remove extra Agrobacterium followed by washing (Fig. 2B3,4); 6. transferring the washed hypocotyls from step - 5 on fresh B₁Ni (BAP and NAA) medium containing antibiotic selection in a container, which is sealed and kept at about 20 to 25°C under cool white fluorescent light for the induction of callus and initiation of root hairs (Fig. 2b'). It is observed that callus initiates within about 3 to 4 days and root hairs initiate from callus mass within about 5 to 10 days (Fig. 2c');

7. start of appearance of green spots in the callus of step — 6. It is observed that after another about 12 to 20 days the green spots start appearing in the callus, and within next about 12 to 20 days, the callus starts appearing granular and often anthocyanin pigmentation is observed in few species of Br assica; 8. transferring the green portion of the callus of step — 7 after another about 1 month period to fresh B₁N₁ medium, which surprisingly results in very fast growth of green spots and within about 10 days these green spots develop into small plantlets (Fig. 2d');

9. sub-culturing the plantlets from step - 8 in a container on a MI (MS + IAA) medium to enhance the root development (Fig.1 e-f.);

10. transferring the plants from step - 9, particularly when the plants show developed root system to the soil after washing the roots thereof;

11. covering the developed plantlets having enhanced roots from step - 10 for hardening (Fig. Ig.); and 12. transferring the hardened plants from step - 12 to the green house after a period of about 12 to about 15 days.

Therefore, in accordance with second embodiment of the present invention, it is possible to deliver gene construct with salt tolerance, drought tolerance, high temperature tolerance, heavy metal tolerance, increase food values, higher yields, disease resistance, pest resistance and the like in varieties of Brassica crop, which could not have been possible with conventional methods.

In accordance with preferred embodiment of this invention, the hypocotyls explants were placed horizontally in a manner that these touch the medium. In accordance with preferred embodiment of this invention, the light chosen is cool white fluorescent light.

In accordance with preferred embodiment of this invention, when the plants show developed root system, they are taken out, the roots are washed with sterile distilled water to remove agar after which, they are transferred to pots, preferably plastic pots containing the soil mixture and are covered, preferably with plastic bags for hardening (Fig. Ig.).

The bag covering the plants are removed before transferring to the green house.

In accordance with preferred embodiment of this invention, the seeds of Brassica sps. are washed with sterile distilled water several times, preferably inside the laminar flow.

In accordance with preferred embodiment of this invention, the treatment of seeds with HgCl₂ solution is carried out for about 5 min.

In accordance with preferred embodiment of this invention, the treated seeds are washed with ethanol for about one minute preferably in a sterile container.

In accordance with preferred embodiment of this invention, the seedlings are removed from the container, which is preferably culture tube employing a pair of sterile forceps under sterile conditions.

In accordance with preferred embodiment of this invention, the hypocotyls are cut with sterile scalpel in a sterile container, which is preferably a petridish.

Therefore, as described hereinabove, an efficient, stable and reproducible regeneration method is disclosed which is capable of improving economical values of the Brassica sps. by enhancing regeneration frequency of the Brassica sps. to produce transgenic plant in one embodiment and genetically modified plant in second embodiment.

As it is clear from the foregoing description that the medium at each of the process step remains same, sequence of process steps also remain same, number of process steps also remains same, and the process parameters also remain same for each of the Brassica sps., accordingly, it may concluded that the method disclosed herein is universally applicable for various varieties of Brassica sps.

The present invention is now described with the help of following examples, which are incorporated merely for elaborating the invention and are not intended to limit its scope. Examples :-

The regeneration frequency of various varieties of Brassica species was improved by employing the present invention. The results of few are tabulated herein below:-

Brassicajuncea

A] Brassica juncea var. ,RH-Sl 9

B] Bmssicαjunceα vax.¹KH.-99Ql

C] Brassicajuncea var.RH-8812

D] Brassicajuncea var.RH-0119

E] Brassicajuncea var.RH-8813

F] Brassicajuncea var.RH-8814

G] Brassica carinata var. HC-209

H] Brassica carinata var. HC-210

I] Brassica napus var. HNS-9605

Claims

J] Brassica nigraIt is clear from the above data that the regeneration frequency is enhanced within a range of 40% to 60% in all different types of seeds of Brassica species.It may be noted that the seeds of various Brassica species are commercially available.Various terms have employed in the specification, which may not be given narrow meaning. However, for the purpose understanding, without prejudice, the term regeneration means a process of growing a plantlet from a single cell or group of cells; the term transformation means the genetic alteration of a cell resulting from the uptake, genomic incorporation, and expression of foreign genetic material (DNA); the term transgenic plant means the transgenic plant possessing a gene or genes that have been transferred from a different species. Although DNA of another species can be integrated in a plant genome by natural processes, the term "transgenic plants" refers to plants created in a laboratory using recombinant DNA technology. The term cole crop is a general term used to describe several vegetables in the mustard family, including broccoli, Brussels sprouts, cabbage, and cauliflower. Claims

1. A method for enhancing the regeneration frequency of varieties of Brassica sps., wherein the method is characterized by comprising treatment of hypocotyls explants segments of Brassica sps. with a medium comprising

Benzyl Amino Purine and Napthalene Acetic Acid [B₁N₁ medium], wherein the treatment results in fast callus initiation, root hairs initiation, appearance of green spots in the callus so formed, and granulation of callus so formed, and appearance of anthocyanin pigmentation in few species of Brassica.

2. A method as claimed in claim 1, wherein the method further comprises treating green portion of the callus after granulation of callus and appearance of anthocyanin pigmentation in few species of Brassica with fresh batch of a medium comprising Benzyl Amino Purine and Napthalene Acetic Acid [fresh batch of BjNi medium], wherein the treatment results in fast growth of plantlets.

3. A method as claimed in claim 1 or 2, wherein the method further comprises treating plantlets of Brassica Sps. with a medium comprising Murashige and Skoog's medium and Indole Acetic Acid [MI (MS + IAA) medium], wherein the treatment results in enhancement of root development resulting in developed plantlets having developed root system, which are capable of growing as hardened plants in the green house.

4. A method for enhancing the regeneration frequency of varieties of Brassica sps., wherein the method comprises treating hypocotyls explants segments of Brassica sps. with a medium comprising Benzyl Amino Purine and Napthalene Acetic Acid [B₁N₁ medium] to result in fast callus initiation, root hairs initiation, appearance of green spots in the callus so formed, and granulation of callus so formed, and appearance of anthocyanin pigmentation in few species of Brassica, followed by treating green portion of the callus after granulation of callus and appearance of anthocyanin pigmentation in few species of Brassica with fresh batch of a medium comprising Benzyl Amino

Purine and Napthalene Acetic Acid [fresh batch Of B₁N₁ medium] to result in fast growth of plantlets, followed by treating plantlets of Brassica Sps. with a medium comprising Murashige and Skoog's medium and Indole Acetic Acid [MI (MS + IAA) medium] to result in enhancement of root development resulting in developed plantlets having developed root system, which are capable of growing as hardened plants in the green house.

5. A method as claimed in any one of the preceding claims 1 to 4, wherein the plant produced is transgenic plant.

6. A method as claimed in any one of the preceding claims 1, 2 or 3, wherein the method further comprises incubating hypocotyls explants segments with a medium comprising Benzyl Amino Purine and Napthalene Acetic Acid [B₁N₁ medium] for check of any infection, and thereafter treating [infecting] with Agrohacterium culture containing the gene of interest on liquid B₁N₁ medium before the treatment of hypocotyls explants segments with B₁N₁ medium, wherein the treatment results in transfer of gene of interest to Brassica sps.

7. A method for enhancing the regeneration frequency of varieties of Brassica sps., wherein the method is characterized by comprises incubating hypocotyls explants segments with a medium comprising Benzyl Amino Purine and Napthalene Acetic Acid [BjN₁ medium] for check of any infection, and thereafter treating [infecting] with Agrobacterium culture containing the gene of interest on liquid B₁N₁ medium, wherein the treatment results in transfer of gene of interest to Brassica sps., followed by treating Agrobacterium treated hypocotyls with a medium comprising Benzyl Amino Purine and Napthalene Acetic Acid [B₁N₁ medium] to result in fast callus initiation, root hairs initiation, appearance of green spots in the callus so formed, and granulation of callus so formed, and appearance of anthocyanin pigmentation in few species of Brassica, followed by treating green portion of the callus after granulation of callus and appearance of anthocyanin pigmentation in few species of Brassica with fresh batch of a medium comprising Benzyl Amino

8. A method as claimed in any one of claims 6 or 7, wherein the plant produced is genetically modified plant.

9. A method as claimed in any one of the preceding claims, wherein the method further comprises steps of:- a). transferring the plants having the developed root system to soil; b). covering the plants of step - a) for hardening; and c). transferring the hardened plants from step — b) to the green house.

10. A method as claimed in any one of the preceding claims, wherein the hypocotyls explants segments are obtained from the hypocotyls of the seedlings incubated in Murashige and Skoog's medium [MSO Medium].

11. A method as claimed in claim 10, wherein the MSO medium comprises Gamborg vitamin.

12. A method as claimed in any one of the preceding claims, wherein the Benzyl Amino Purine is 6-Benzyl Amino Purine.

13. A method as claimed in any one of the preceding claims, wherein the callus initiation and root hairs initiation start within a period varying upto about 15 days from the day of start of treatment.

14. A method as claimed in claim 13, wherein the callus initiation starts within a period varying from about 3 to about 5 days from the day of start of treatment, and root hairs initiation starts within a period varying from about 5 to about 10 days from the callus mass so formed.

15. A method as claimed in any one of the preceding claims, wherein the appearance of green spots in the callus mass having root hairs starts within a period varying from about 12 to about 20 days after the root hairs initiation in the callus.

16. A method as claimed in any one of the preceding claims, wherein the callus starts appearing granular in another period varying from about 12 to about 20 days after appearance of green spots, and formation of anthocyanin pigmentation in few species of Brassica Sps. starts simultaneously to granulation of callus mass.

17. A method as claimed in any one of the preceding claims, wherein the development of small plantlets starts within a period varying upto about 10 days from start of treatment with fresh batch OfB₁N₁ medium.

18. A method as claimed in any one of the preceding claims, wherein the treatment with B₁N₁ medium is carried out under light, preferably cool white fluorescent light.

19. A method as claimed in any one of the preceding claims, wherein the hypocotyls are cut into segments having a length of about 5 to 7 mm.

20. A method as claimed in any one of claims 6 or 7, wherein the hypocotyls being treated with Agrobacterium culture are simultaneously treated with antibiotic solution-augmentin.

21. A method as claimed in any one of claims 6, 7 or 20, wherein the treatment with B₁N₁ medium is carried out in presence of antibiotic solution.

22. A method as claimed in any one of the preceding claims, wherein the hypocotyls of about 5 to 6 days old seedlings are taken, and wherein the hypocotyls taken have a length varying from about 4 to 5 cm.

23. A method as claimed in any one of the preceding claims, wherein the hypocotyls are cut into hypocotyls explants segments of a length varying from about 5 to about 7 mm.

24. A method as claimed in any one of the preceding claims, wherein the B₁N₁ medium comprises Benzyl Amino Purine and Napthalene Acetic Acid, which are taken in equal amounts, preferably in an amount of about 2mg per L.

25. A method as claimed in any one of the preceding claims, wherein in MI medium each of Murashige and Skoog's medium and Indole Acetic Acid are taken in equal amounts, preferably in an amount of about 2mg per L.

26. A method as claimed in any one of the preceding claims, wherein the hardened plants are transferred to the green house after a period of about 12 to about 15 days.

27. A method as claimed in any one of the claims 6, 7, 20, or 21, wherein the strains of Agrobacterium tumefaciens is one which is capable of transferring genetic material to Brassica species, preferably it is A. tumefaciens strain

GV3101.

28. A method for enhancing the regeneration frequency of varieties of Brassica sps. substantially as herein described with the help of foregoing examples and as illustrated in the accompanying figures.