Optimization of rhizogenesis for in vitro shoot culture of Pinus massoniana Lamb.

The rooting capacity of Pinus massoniana is poor,especially for mature trees,and has prevented the development of clonal forestry for P.massoniana.In this study,we varied explant types,subculture times and exogenous hormones for plantlet regeneration and assessed shoots for rooting rate and root number for P.massoniana.Following fi ve repetitive grafts,new shoots from grafts used as explant sources were rejuvenated as observed from juvenile shoot morphology and anatomy,leading to greatly enhanced plant regeneration in comparison to that of mature materials from 26-year-old P.massoniana trees.The rooting capacity of subcultured shoots increased with successive subcultures,reaching a peak at 20 subcultures with 35–40 days per subculture.However,rooting performance was signifi cantly reduced after 30 subcultures.The addition of naphthaleneacetic acid(NAA)plus indoleacetic acid in the medium improved the root number,but the combination of exogenous NAA with paclobutrazol(PBZ)increased rooting rate and root number.We thus greatly improved the rooting capacity of mature P.massoniana trees by optimizing explant types(rejuvenated),subculture times(20 subcultures,35–40 days per subculture)and addition of NAA+PBZ to the rooting medium.The conditions can be used for effi cient plantlet regeneration of P.massoniana.

Rhizogenesis of Two Species Fabaceae: <i>Cicer arietinum</i>L. and <i>Pisum sativum</i>L.

Our work of comparative study on the biomorphological, rhizogenic level of Chickpea (Cicer arietinum) and Pea (Pisum sativum), includes successively the following results: The in-vitro culture done, showed strains, which can reach 6.5 mm in the medium Nutrient agar (NA) for Cicer arietinum 6 mm of strain were recorded in the PDA culture medium for Pisum sativum. The best strain is obtained in temperature of 20&#8451;(ambient). According to the results of the rhizogenesis, we notice that the nutrient agar (NA) medium responds better that the PDA medium for Cicer arietinum. Meanwhile, the medium PDA brings a better reaction (response) compared with the nutrient agar medium concerning Pisum sativum.

Changes of phenolic compounds in Carignan merithallus (Vitis vinifera L.) during bud dormancy and end of dormancy phase: correlation with rhizogenesis

The aim of the present study is to address the type of correlation that may exist between phenolic compounds and vine rhizogenetic potential by analyzing some phenolic compounds in the Carignan merithallus. Phenolic compounds were analyzed by HPLC in the young shoots (or merithallus) of Carignan vine (Vitis vinifera L.) and we established a correlation between the studied compounds and the rhizogenetic potential of shoots during the phases of bud dormancy and end of dormancy, taking into account the position effect on shoots. This work was carried out for the first time on this type of vine. Among the studied phenolic compounds, we observed a negative correlation between coumarin and rhizogenetic potential of Carignan vine. In contrast, positive correlations were found with naringin and syringic acid. Obtained results confirmed the fact that the action of phenolic compounds is complex and might be qualified as cofactors that interact with auxin on rhizogenesis.

Plant Regeneration through Indirect Organogenesis in Two Cultivars of Pineapple (Ananas comosus L.)

Unavailability of performant planting material of pineapple constitutes a major problem of its cultivation in Africa. For this purpose, indirect organogenesis technique is used to evaluate the in vitro responses of two cultivars of pineapple during the explant’s regeneration. Calli were induced from crown leaf and plantlets leaf of “Smooth Cayenne” and “Sugarloaf cultivars”. Murashige and Skoog medium with vitamins B5 supplemented with different growth regulators combinations were used. BAP and/or 2,4-D have been added to base medium for calli cells’ differentiation while BAP and GA3 have been added for plant elongation. The results indicated that explants from regenerated plantlets leaves cultivated on MS supplemented with copper (II) sulphate 5-hydrate concentrations incorporated had significant (p < 0.0001) influence on callus induction in pineapple cultivars. Likewise, MS medium with NAA (0.5 mg/l) + BAP (1 mg/l) had a highly significant influence with 8.8 differentiated Calli. Also, MS medium supplemented with BAP (3 mg/l) + GA3 (2 mg/l) for the “smooth Cayenne” had significantly influenced (p < 0.0001) Calli regeneration with a high rate of 55.25% plantlets. MS medium containing 0.5 mg/l of NAA + 0 mg/l IBA produced a high number of roots in Sugarloaf whereas the medium containing 1.5 mg/l NAA + 0.5 mg/l (IBA) produced high number of roots in smooth Cayenne. We have established an efficient and reproducible protocol for mass propagation and genetic transformation of pineapple though indirect organogenesis. This protocol may be used in genetics engineering studies for pineapple breeding.

Low temperature,IBA concentrations and optimal time for adventitious rooting of Eucalyptus benthamii mini-cuttings

Eucalyptus benthamii is a forest species of economic interest that has difficulty with seed production and also is considered to have difficulty with adventitious rooting using propagation techniques, such as cutting or mini-cutting. We aimed to assess the adventitious rooting percentage under different storage times in low temperatures and at various IBA （indole-3-butyric acid） concentrations to determine the optimal time of permanence for rooting Eucalyptus benthamii minicuttings in a greenhouse. Shoots collected from mini-stumps cultivated in a semi-hydroponic system were used to obtain the mini-cuttings. For the first experiment, the mini-cuttings were stored at 4℃ for 0 （immediate planting）, 24, 48, 72, 96 and 120 h. The second experiment evaluated the rooting dynamic to determine the optimal time of permanence for minicuttings in a greenhouse. The basal region of the mini-cutting was treated with various IBA solutions： 0 （free of IBA）, 1,000, 2,000, 3,000 and 4,000 mg.L^-1. Every seven days （0 （immediate planting）, 7, 14, 21 and 28 days）, destructive sampling of the mini-cuttings was performed to evaluate the histology of the adventitious rooting. Eucalyptus benthamii minicuttings should be rooted immediately after the collection of the shoots. The 2,000 mg.L^-1 IBA concentration induced a greater speed and percentage of adventitious rooting, and an interval of 35 to 42 days was indicated for permanence of the mini-cuttings in the greenhouse. Expo- sure to low temperature induced adventitious root formation with diffuse vascular connections.

Efficient Evergreen Plant Regeneration of Cinnamomum japonicum Sieb.through in vitro Organogenesis

Cinnamomum japonicum Sieb.is an excellent roadside tree and medicinal tree species with considerable ornamental and economic value.In this study,we successfully developed a large-scale micropropagation protocol for C.japonicum for the first time.Sterilized shoots were excised and used as explants for shoot induction on several basal media,supplemented with different concentrations of plant growth regulators(PGRs),such as Thidiazuron(TDZ),N^(6)-Benzyladenine(6-benzylaminopurine)(BA),α-naphthaleneacetic acid(NAA)and Gibberellic acid(GA_(3)).After comparison,the most efficient medium for shoot regeneration was 1/2 Murashige and Skoog(MS)medium containing 0.5 mg L^(-1)BA,0.05 mg L^(-1)NAA and 0.2 mg L^(-1)GA_(3),which resulted in an average number of induced shoots per explant and shoot length of 5.2 and 1.62 cm at 28 d,respectively.Then,elongated adventitious shoots were transferred to induce roots.86.7%of shoots was able to root on 1/2 MS medium supplemented with 0.5 mg L^(-1)NAA and 0.1 mg L^(-1)BA.The earliest rooting time observed was after 21 d and the average root length was up to 3.3 cm after 28 d.Our study shows that C.japonicum can be successfully regenerated through de novo organogenesis,which lays a foundation for future transformation research on this tree.

<i>In vitro</i>propagation of <i>Aegle marmelos</i>(L.) corr., a medicinal plant through axillary bud multiplication

A protocol for rapid in vitro propagation from nodal explants of the medicinal tree species Aegle marmelos (L.) corr. of family Rutaceae has been described. High frequency bud break were induced on Murashige and Skoog’s (1962) basal medium supplemented with 0.5 mg benzyladenine (BA)/l. After 10 days of culture, nodal explants with multiplied buds started callusing with restricted growth and defoliation. When the same nodal explants ware transferred into the same basal medium supplemented with 0.5 mg BA/l with different concentrations of either kinetin (KN) or gibberellic acid (GA3) or in combinations has shown healthy shoots with expanded shoot length. Excised shoots (2cm-3cm long with 2 to 3 nodes) when grown on 1/2 MS basal medium with 2.5 mg/l indole-3-butyric acid (IBA) and 0.5% activated charcoal (A.C.)/l has shown rhizogenesis. After excision, in the second passage, the nodal explants also showed bud break when sub cultured on MS basal medium supplemented with 0.5 mg BA/l. These shoots also successfully rooted on the same above said medium.

Influence of Exogenous Auxins on Phenolic Compounds Contents and Polyphenol oxidasic and Peroxidasic Activities in Root Differentiation in <i>Gnetum</i>spp.

Root failure is a key limiting factor in Gnetum spp. domestication and production of plants materials for farming. To overcome, root failure in Gnetum spp. may depend on adequate strategies set up and understanding of biochemical markers controlling the rooting process. The present work is principally based on phenolic compounds contents, peroxidasic and polyphenol oxidasic activities during rooting of cuttings (pretreated with IBA, ANA and IAA) at induction, initiation and expression phases of roots formation in Gnetum africanum Welw. and G. buchholzianum Engl. The study revealed that cuttings pretreated with IBA (150 mg/l) were more effective than IAA and NAA. Phenolic compounds analysis revealed that cuttings pretreated with IBA exhibited high phenolic compound contents at initiation and expression phases for both Gnetum species. Indicating that IBA pretreatment promotes phenolic compounds synthesis and accumulation necessary for roots differentiation. Enzyme contents appear to be high during the initiation stage for both species when they are pretreated with IBA (150 mg/l). Peroxidasic and Polyphenol oxidasic activities evolved on the synchroneous maner. This allows us to know that the implication of these enzymes in the rooting process is fundamental makers.

Assessing the response of indigenous loquat cultivar Mardan to phytohormones for in vitro shoot proliferation and rooting

In vitro cultures of loquat cultivar Mardan were established using shoot apices after treating with NaOCl (5%,7%,10%,12%,14% (v/v)) for 12 min and HgCl2 (0.01%,0.05%,0.10%,0.20%,0.25% (w/v)) for 2 min.A maximum survival rate of 70% was recorded after surface sterilization with 10% NaOCl.Caulogenic response was assessed on Murashige and Skoog (MS) medium fortified with assorted combinations of the cytokinins,benzylaminopurine (BAP),kinetin,and N6-(2-isopentyl)adenine (2iP).Treatment of BAP 1.5 mg/L combined with 2iP 9.0 mg/L and kinetin 1.5 mg/L was found to be optimum for shoot morphogenesis in terms of the number and subsequent growth of shoots,while the highest shoot length was yielded by the combination of BAP 0.5 mg/L,kinetin 0.5 mg/L,and 2iP 3 mg/L.Higher levels of cytokinins induced callogenesis,vitrification and stunted growth to some extent.For rhizogenesis,uniform sized micro-shoots were excised and transferred to half-strength MS medium containing auxins.The best rooting expression was observed with naphthaleneacetic acid (NAA) 1 mg/L combined with indole-3-butyric acid (IBA) 2 mg/L and paclobutrazol (PBZ) 1 mg/L.