Induction of callus and somatic embryogenesis from explants of Parkiabiglobosa ( Jacq . ) Benth

Legumes are very important species including herbs, shrubs and trees, they are cultivated worldwide for different economic uses.Parkiais genus belongs to the Legumefamily,Fabaceae. The most common known species are P. biglobosaBenth (African locust bean) and P. roxburghii (Tree Bean)which were introduced to Egypt since 1890. The population of the tree is rapidly declining as result conservation efforts is needed to prevent it from extinction. In an attempt to conserve this genetic resource, tissue culture studies were carried toestablish a callus culture of both plants from different explants and regeneration of the developed callus and to study the effect of explant type, media composition on the callus production and the regeneration. Apical bud,axillary bud and root explants from in vitro germinated seedlings of P. biglobosaBenthwere cultured on MS and B5 medium supplemented with (0.4., 0.8, 1.2, 1.6 or 2.0 mg l -1 NAA) + Glutamine 0.5 mg l -1 + Proline 0.5 mg l -1 + Casein 0.3 mg l -1 + 2,4D 2.5 mg l -1 . Each medium was supplemented with 30 g l -1 sucrose, 3 g l -1 agar and PH 5.8.for callus induction .and (0.4.,0.8,1.2,1.6 or 2.0 mg l -1 NAA) + Glutamine 0.5 mg l -1 + Proline 0.5 mg l -1 + Casein 0.3 mg l -1 + 6-BAP 2 mg l -1 . Each medium was supplemented with 30 g l -1 sucrose, 3 g l -1 agar and PH 5.8.for regeneration.The study presented here demonstrates the successful attempt at regeneration of plantlets of P. biglobosaBenth via indirect organogenesis.It can be concluded that the medium composition has a significant effect on the calli production together with the type of the explant which showed relatively higher significance. Seeds germination stage apical bud explants media 8 followed by media 10 showed the most favorable mean performances values for the studied characters, number of days to callus initiation (day), callus weight (mg), dead calli percentage (%), albino plants percentage (%),green plants percentage (%) and root percentage (%). Moreover, axillary bud, showed remarkable increase in the mean performance of average. However, the overall response of MS medium was found to be superior to that of B5 medium. Explants cultured on MS medium fortified with combinations of 2,4-D and BAP induced rapidly proliferating calli that turned more friable and nodular. Indexing terms/


ABSTRACT
Legumes are very important species including herbs, shrubs and trees, they are cultivated worldwide for different economic uses.Parkiais genus belongs to the Legumefamily,Fabaceae.The most common known species are P. biglobosaBenth (African locust bean) and P. roxburghii (Tree Bean)which were introduced to Egypt since 1890.The population of the tree is rapidly declining as result conservation efforts is needed to prevent it from extinction.In an attempt to conserve this genetic resource, tissue culture studies were carried toestablish a callus culture of both plants from different explants and regeneration of the developed callus and to study the effect of e xplant type, media composition on the callus production and the regeneration.Apical bud,axillary bud and root explants from in vitro germinated seedlings of P. biglobosaBenthwere cultured on MS and B5 medium supplemented with (0.4., 0.8, 1.2, 1.6 or 2.0 mg l -1 NAA) + Glutamine 0.5 mg l -1 + Proline 0.5 mg l -1 + Casein 0.3 mg l -1 + 2,4D 2.5 mg l -1 . Each medium was supplemented with 30 g l -1 sucrose, 3 g l -1 agar and PH 5.8.for callus induction .and(0.4.,0.8,1.2,1.6 or 2.0 mg l -1 NAA) + Glutamine 0.5 mg l -1 + Proline 0.5 mg l -1 + Casein 0.3 mg l -1 + 6-BAP 2 mg l -1 . Each medium was supplemented with 30 g l -1 sucrose, 3 g l -1 agar and PH 5.8.for regeneration.The study presented here demonstrates the successful attempt at regeneration of plantlets of P. biglobosaBenth via indirect organogenesis.It can be concluded that the medium composition has a significant effect on the calli production together with the type of the explant which showed relatively higher significance.Seeds germination stage apical bud explants media 8 followed by media 10 showed the most favorable mean performances values for the studied characters, number of days to callus initiation (day), callus weight (mg), dead calli percentage (%), albino plants percentage (%),green plants percentage (%) and root percentage (%).Moreover, axillary bud, showed remarkable increase in the mean performance of average.However, the overall response of MS medium was found to be superior to that of B5 medium.Explants cultured on MS medium fortified with combinations of 2,4-D and BAP induced rapidly proliferating calli that turned more friable and nodular.

INTRODUCTION
Parkia is a genus of about 30-40 tree legume species of considerable evolutionary, ta xonomic, biological and economic importance in Africa, Asia and South America.It has several uses, including fodder, food, medicine, green manure, fuel and timber [1].
Parkiaroxburghii G. Don.isknown as "tree bean, It is a multipurpose tree with intermediate height (10-20 m) the tree known to be highly branched.The green pods and seeds ofParkiaroxburghii have a high nutritional value and can serve as a potential source of protein and fat and also used in various local medicinal applications [2,3].P. biglobosa is an important tree species which generates non-timber forest products popularly known as the African locust bean tree belongs to the family Fabaceae [4,5,6].It is a perennial deciduous tree occurring in a belt between 5° N and 15° N [6], 7 to 20 m tall, and in some cases it can reach up to 30 m [7].It is considered to be valuable source of nutrition and as therapeutic food [4].The pulp of the fruit pods is rich in sucrose and the seeds are rich in carbohydrates, proteins and lipids, thus constituting an important source of energy [4].P. biglobosa is rated fifth important among thirty-one woody medicinal plants used in traditional medicine in Benin.It is rated fourth from a list of eighteen priority food woody plants to preserve.In association with crops, the species help to enrich physico -chemical soil characteristics which in turn help to increase crop yields [4].The fruit is a slightly curved, brown indehiscent pod, 30 to 40 cm long and 2 to 3 cm wide producing up to 20 seeds.The seeds when boiled and fermented is known as "dawadawa" in Hausa language in Nigeria, a black strong smelling tasty seasoning, rich in lipid 29 per cent, protein 35 per cent, carbohydrate 16 per cent, and it is a good source of fat and calcium for rural dwellers [8].The pods are used as sponges and strings, dyes, and for fishing, and also for preparing insecticide powder [6].The bark is used as a mouthwash, vapour inhalant for toothache, or for ear complaints.It is macerated in baths for leprosy and used for bronchitis, pneumonia, skin infections, sores, ulcers, and washes for fever, malaria, diarrhea, and sterility.Roots are used in a lotion for sore eyes [9].
There are fewssources of these two trees (Parkiabiglobosa and Parkiaroxburghii ) in Egypt which contains the genes of adptability to the Egyptian environment,as results efforts is needed to conserve and prevent theseindigenous plants from extinction .Vegetative propagation by means of tissue culture techniques is an important tool for plant germplasm conservation and rapid clonal multiplication as well as for reforestation and tree improvement.
Micropropagationand tissue culture studiesof Parkiaroxburghii G. Don are still not present and need efforts to establish a good protocol for plant regeneration .Also, tissue culture studies in ParkiabiglobosaBenthare rare and obtaining shoots are still challenging, the literaturesare confined to callus and embryo induction without shoot or plantlet regeneration [10,11] or in vitro germination and multiplication of a P.biglobosa [11].Plant regeneration has been successful in some non-woody horticultural and woody plants [12,13].Reviewing the current literature, it was found that a trial was made to study the effect of plant growth regulators and different media on micropropagation of P.biglobosa (Jacq.)Benthand P. roxburghiiG.Don.A study showed a method for differentiation of callus, shoots and plant regeneration from hypocotyl explants ofParkiabiglobosaBenth via indirect organogenesis.This work aimed to establish a callus culture of both plants from different explants and regeneration of the developed callus and to study the effect o f explant type, media composition on the callus production and the regeneration of the studied plants.
Tissue culture involves the isolation of cells or tissues and placing them under controlled, aseptic environments [14,15,16]).All of the environmental factors necessary for growth (heat, light, air, water, nutrients, and support) are provided artificially with the objective being to obtain rapid asexual multiplication of plant cells or plants.Any given tis sue composed of cells with competent nuclei is a suitable explant for the initiation of a plant tissue culture [17,18].Multiplication of plant materials in vitro involves the manipulation of plant growth through modifications of the culture medium, culture environment, and the source and type of tissue taken for culture [16,18].Multiplication can be achieved by one of three different morphogenicprocesses:axillary bud enhancement, adventitious shoot forma tion, or somatic embryogenesis [14,16,18].

Plant materials
Trees of Parkia.biglobosa and Parkiaroxburghii G. Dongrown in Alzohria garden Giza,Egypt, were used as source of fresh tissue explants for in vitro culture techniques .A trial was made to produce callus by in vitro culturing of different fresh parts of the conventional plant on tissue culture media .Various types of 1-2 cm length explants {apical bud, axillary bud, and leaflet and midrib at the node (nodule explant)} from each parental species P. roxburghiiG.Don.andP.biglobosa (Jacq.)Benth were used.Explants were washed thoroughly with running tap water for 7 min., then dissected, and then wrapped in sterilized lawn in sterilized Petri dish.70 % ethanol (v/v) was used for surface sterilizing of every e xplant for 30 seconds.Every explant was soaked in 40 % Clorox (sodium hypochloride 5.25 %) for 20 min.After 20 min., Clorox was discarded and sterilized explants were washed by sterilized distilled water three times (3 min.each).The authors want to notify that lower concentrations of Clorox (10, 20 and 30%) which were used as trials for sterilizing the explants have been failed to supply sterile conditions.
Matured seeds of P. biglobosa Jack.Benth.Were obtained from Germplasm Resources Information Network (GRIN), USDA and used to develop seedling as source of fresh young explants (apicalbuds, axillary buds and roots).In order to chemically break the seed dormancy and reduce the testa toughness using 70% H 2SO4 for 20 min.Surface sterilization was done by treating with 1% (v/v) sodium hypochlorite for 15 min, a nd rinsed three times with sterile distilled water.Seeds were then germinated on MS medium, with 10 g/l sucrose and 8 g/l agar in a growth room at 25±1 ℃, 16 h photoperiod, 30-40µmol m-2 s-1 cool white fluorescent light.The pH of the medium was adjusted to 5.8 prior to the addition of agar and autoclaving at 121℃ for 15 min.The obtained in vitro seedlings (2-6 weeks old) were used as the source of the explants in the induction of callus.
Three different fresh explants types (apical, axillary buds and roots) from 2-5 weeks old seedlings of P. biglobosawere cut with a fine sterile blade to produce small pieces of about (10 mm x 10 mm), the explants pieces were cultured in Petridishes containing 33 ml of callus induction medium.For each medium type, 3 replications were done, each replication with 2 Petri-dishes, and each Petri-dish contained 10-15 explants of a particular accession.An a verage of 160 explants (by repetition ) per cultivar were used in the experiment.The cultures were maintained in dark at 25±2ºC in dark with a relative humidity of 60%.All cultures were placed at 25±1℃, 16h photoperiod, 30-40 µ mol m-2 s-1 cool white fluorescent light.All the cultures were sub cultured at two weeks intervals on fresh media.Callus induction was visually evaluated and scored to aid a more rapid screening option.The explants were sub cultured on freshly prepared media at two weeks intervals until callus developed.The chemicals used for the experiments were manufactured by M/S HimediaCompany,Mumbai, India.

Callus growth and regeneration ability characters:
Observationswere recorded on three plates taken at random from each medium used for each test entry for determination of the following criteria:-1.Number of days to callus initiation (It was counted as a number of days from date of plating to the date of first callus appear in first three plates).2. Percentage of callus induction ability (It was calculated as apercentage of induced calli per anthers in a plate).3.Green plants percentage (%) (It was calculated as percentage of green plants regenerated from calli).4. Albino plants percentage (%) (It was calculated as percent of albino plants regenerated from calli).5.Dead calli percentage (%) (It was calculated as percentage of calli which did n ot regenerate, dead calli have brown color and do not show any differentiation).6.Root percentage (%)It was calculated as percentageof roots which developed from calli and did not show any shoot differentiation .

Statistical analysis
The experimental design was a completely randomized design with three replications per medium type, 10 -15 explants per accession per replicate.For the experiment on callus induction, 3 accessions, 3 types of explants at 4 types of media were analyzed as a 3 x 3 x 4 factorial arrangement.In this study, callus from leaf explants did not regenerate shoots, therefore, the analysis on regeneration were based on the number of hypocotyls explants cultured.Hence, 3 accessions at 4 types of media were analyzed as a 3 x 4 factorial arrangement.Data obtained were subjected to analysis of variance (ANOVA).Significant means were separated using least significant difference (LSD) test.All percent data were subjected to arc sine (√x) transformation before statistical analysis.

Induction of callus from the different plant organs of the conventional plant
The effect of e xplant types and plant growth regulators on the in vitro callus formation was studied and the callusing capacities, as well as, the types of explants on the different media were recorded in and tables (2,3and 4) and fig.
(1,2,3).The mean performance of the different criteria of all explant for the two studied species is presented in tables 3 -5.
All the regeneration produced from different media and variable explants were evaluated.

Number of days to callus initiation (day):
The obtained outcomes of mean performance characters of P. biglobosa (Jacq.)Benth, table (2) illustrated the shortest mean period to number of days to callus initiation for the apical bud explant (28.31) in media 10 followed by the axillary bud explant (29.55) in media 8. While, the nodule explan t (51.39) in media 2 scored the largest number of days.On the other hand, P. roxburghiiG.Don.recorded the shortest mean period to number of days to callus initiation for the apical bud explant (28.33) in media 8 followed by the axillary bud explant (29.88) in media 10 While, the nodule explant (58.33) in media 5 scored the longest period (Table 2).

Dead calli percentage (%):
On comparing the dead calli percentage % it was cleared that the mean performance characters of P. biglobosa (Jacq.)Benth, showed thelowest percentage of dead callifor the apical bud explant (31.68) in media 8 followed by the axillary bud explant (33.71) in media 2. While, the nod ule explant scored the highest percentage (71.25) in media 7.Meanwhile, the mean performance characters of P. roxburghiiG.Don.Showed the lowest percentagefor the apical bud explant (31.17) in media 8 followed by the axillarybud explant (32.58) in media10.While, the nodule explant scored the highest percentage of dead calli (70.35) in media 10(Table 4).
Theresults as shown in tables 2, 3 and 4 recommended that media 2, 3,8 and 10 as they were the most suitable media for callus production as revealed from the three mean perform ance of the measured factors (number of days to callus initiation (day), callus weight and dead callus percentage).Concerning explants, apical bud and axillary bud showed appropriate callus result as demonstrated in the previous results.That"s why we decide to continue using those explants from the germinated seeds.Accordingly to these results in our present study we decided to continue using those explants from the germinated seeds.

Induction of callus from the in vitro germinated seedlings:
The mean performance of selected parts as source of explant from P. biglobosa(Jacq.)Benth are presented intables 5, 6 and all.theregeneration formation were evaluated.

Number of days to callus initiation (day):
The obtained outcomes of mean performance characters of P. biglobosa (Jacq.)Benthillustrated the shortest mean period of number of da ys to callus initiation (day) for the apical bud explant (28.363) media 8 followed by the root explant (30.557) media2.On the other hand, the axillary bud e xplant (36.207) media 2 showing the longest period as shown in table 6.

Callus weight (mg)
It could be concluded from table 6 that, the mean performance characters of P. biglobosa (Jacq.)Benthdemonstrated the highest m ean performance of callus weight (mg),for the apical bud explant (0.683) media 8 followed by media 3 (0.623) for the same explant.On the other hand, the a xillary bud explant (0.459) media 2 scored the lowest callus weight (mg).Green plants percentage (%)

DISCUSSION
Tissue culture studies on s eeds of P. biglobosaBenth to prevent it from extinction.Leaf and hypocotyl explants from in vit ro germinated seedlings were cultured on MS medium containing different concent rations of 6-benzylaminopurine (BA) either alone or in combination with (NAA ) for callus induction.Seeds produced the highest percent age (80%) of callus than from leaf explants.[10] and in vit ro conservation of P. biglobosain Senegal through tissue culture technique.Different concent rations of growth regulators were added alone or in combination in a MS basal medium.During acclimatization ac hievement, survival rates were respectively 80% for apex and explants and 86.66% for axillary explants.

CONCLUSION
The study presented here demonstrates the successful attempt at regeneration of plantlets from apical and axillary buds together with root explants of P. biglobosaBenth via indirect organogenesis.Further studies are needed to increase the regeneration frequency and to induce shoots from other explants.Our results suggest that this protocol would provide useful information for mass propagation and germplasm conservation , and would help in the further work concerning genetic transformation to incorporate useful genes and for the production of valuable secondary metabolites.
It can be concluded that the medium composition has a significant effect on the calli production together with the type of the explant which showed relatively higher significance.Medium 3, 8 and 10 gave the highest yield of the cultured callus.Concerning the types of explants, the most viable explants used for callus production and for further regeneration were apical and axillary buds together with root explants .

Fig 1 :, 3 and 8 .Fig 2 :
Fig 1: show s plant regeneration through callus formation ( callus of the different explants).A Callus of leaflet and node explants on media2, 3 and 8. Two week old.B callus of axillary bud explants on media 2, 3 and 8.Four week old callus.Callus of apical bud Six week old callus on media 2, 3 and 8.

Fig 4 :Fig 5 :Fig. ( 6 )
Fig 4: Mean performance of P. biglobosa explants to Days to callus initiation on the used media

Fig 8 :Fig 8 :
Fig 8: Mean performance of P. biglobosa explants to green plants percentage %on the used media

[ 11 ]
.Cotyledon explants of P. timoriana for in vitro rapid regeneration, on MS and B5 bas al media supplemented with various concent rations of 2, 4-D, NAA and BAP.Successful callus induction was observed in all the treatments.Maximum percentage of callus induction was obtained in the 2, 4 -D supplement ed basal media.Explants cultured on MS medium fortified with combinations of 2, 4-D and BAP.[3]

Table 2 .Mean performance of parental genotype s or species, mediums and explant for number of days to callus initiation (day).
LSD: Least significant difference at 1%.

Dead Plant species P. biglobosa Criteria Dead calli percentage % Albino plants percentage %
table 48, for the apical bud e xplant (24.473) media 8 followed by the axillary bud explant (27.047) in the same media.Meanwhile, the axillary bud e xplant (41.440) media 3 scored the highest mean performance of dead calli percentage.asshown in table 6. Fig.6Data in table 48, proved that the lowest mean performance characters percentage of Albino plants, wasfor the apical bud e xplant (11.40) media 8 followed by the root explant (19.723) media2 while, the apical bud explant (39.573) media 10 scored the highest percentage of Albino plants.asshown in table 6. Fig.7Considering the mean performance characters of the studied plant, it was illustrated that the highest percentage of green plants was for the apical bud explant (61.750) media 8 followed by the root explant (57.120) media10.On the other hand, the axillary bud explant (22.413) media 2 scored the lowest percentage of green plants as shown in table7, fig 8.Moreover, axillary bud, showed remarkable increase in the mean performance of average.However, the overall response of MS medium was found to b e superior to that of B5 medium.Explants cultured on MS medium fortified with combinations of 2,4-D and BAP induced rapidly proliferating calli that turned more friable and nodular.These results are in accordance with those reported by [ Root percentage (%)It is obvious that, the mean performance characters ofP.biglobosa (Jacq.)Benthrecorded the highest percentage of root for the apical bud explant (53.140) media 8 followed by the axillary bud explant (49.513) in the same media.At the same time, the axillary bud explant (27.373) media 2 showed the lowest percentage of root as shown in table 7, Fig.9.Generally, Seeds germination stage apical bud explants media 8 followed by media 10 showed the most favorable mean performances values for the studied characters, Number of days to callus initiation (day) , Callus weight (mg), calli percentage (%),Albino plants percentage (%),Green plants percentage (%) and Root percentage (%).