Agrobacterium tumefaciens-Mediated Genetic Transformation of Green Gram [Vigna radiata (L.) Wilczek] - A Recalcitrant Grain Legume

Background: Green gram is grown in many parts of India as a source of dietary protein (21-25%). It is an important nitrogen fixing crop which fixes atmospheric nitrogen (119-140 kg/ha) to soil and enhance the soil productivity. In the present investigation, efficient Agrobacterium-mediated genetic transformation of Vigna radiata L. (Wilczek) has been achieved with VrTIP1 gene for abiotic stress resistance i.e. moisture and salinity stress. Methods: Four days old shoot tip and cotyledonary node were used for in vitro regeneration with MS medium supplemented with BAP 2.0 mg/l, kinetin 0.5 mg/l and 50 mg/l kanamycin for co-cultivation with Agrobacterium tumefaciens strains, LBA 4404. The modified binary vector pCXSN, EHA105 containing hygromycin phosphotransferase II (hpt II) marker genes and a synthetic TIP1 gene under a constitutive CaMV35S promoter were used for transformation of Vigna radiata L. cotyledonary node explants. Putative transformants selected from hygromycin resistant shoots were subsequently rooted on MS medium supplemented with 1.0 mg/l NAA and later transferred to sterile vermiculite followed by transfer to the transgenic green house. Result: The T1 plants were produced from PCR positive T0 plants and analysed for presence and integration of transgenes in putative T1 plants were confirmed by polymerase chain reaction (PCR) amplification of 752 bp of hpt II fragment. This protocol can be effectively used for transferring new traits in greengram and other legumes for their quantitative and qualitative improvements.

Effect of Agrobacterium co-cultivation stage on explant response for subsequent genetic transformation in Soybean (Glycine max (L.) Merr.)

The establishment of an efficient in vitro genetic transformation protocol in soybean depends upon an effective interaction between the explants and Agrobacterium tumefaciens. Therefore, a study was conducted at the University of Limpopo, South Africa, between September 2019 and May 2020 to evaluate explant amenability and effects of Agrobacterium co-cultivation stage on the induction of oxidative stress. This stress potentially causes lipid peroxidation, reduction of phytochemicals and chlorophyll pigments on explant tissue targeted for genetic transformation. This study, used double cotyledonary node explants infected and co-cultured with A. tumefaciens to evaluate total phenolics, antioxidant activity, lipid peroxidation and oxidative stress-induced tissue senescence during the co-cultivation stage. The results, showed that, explant co-cultivation with Agrobacterium for 2, 4 and 6 days caused reductions in the amounts of phenolic compounds, chlorophylls and antioxidant activity due to tissue senescence, high oxidative stress and malondialdehyde contents. Percentage phenolic content of all bacteria infected explants ranged between 10.3?10.6 compared to 20.9% in the control. Chlorophyll content of about 1.49?4.00 mg/ml and malondialdehyde content ranging between 1.0?5.7 mM-1g-1 were also recorded. Overall, findings suggest that the infection of explants with A. tumefaciens can induce oxidative stress and tissue senescence depending on the period of co-cultivation. However, reduced oxidative stress and senescence of explant tissues may potentially improve soybean shoot regeneration and transformation efficiency.

Rapid Micropropagation Protocol of Mucuna pruriens var. utilis using Cotyledonary Node Explant: A Cultivated Medicinal Edible Legume

Background: The presence of L-dopa coupled with rich protein and amino acid marked Mucuna pruriens var. utilis as an important under-utilised legume. Therefore, it is useful to develop a method for large-scale multiplication for commercial production. Method: Tissue culture technology is successfully utilized in propagation of plants with poor and uncertain response to conventional propagation. Murashige and Skoog’s (MS) medium without any Plant Growth Regulators (PGRs) was used for seed germination and with PGRs for shoot and root multiplication.Result: Highest 95% seed germination was found in fresh seeds at 7-8 days of culture. Shoot multiplication percentage was found to be 100% with highest c.a. 21.1 shoots with an average 4.8 cm shoot length on MS + BAP 1.5 mg L-1 per 10 days old cotyledonary node explant. A total c.a. 144 shoots were harvested after 3rd harvest of the mother cotyledonary node with two whole cotyledons at day 70. Rooting was best induced in in vitro derived shoots on MS medium without any PGRs and plantlets were acclimatized in sand and soil (1:1) and established in pot with garden soil.

In vitro shoot regeneration in Myracrodruon urundeuva Fr. All

ABSTRACT Myracrodruon urundeuva Fr. All. is a tree threatened with extinction, which has wood and medicinal potential. This study aimed to analyze the in vitro shoot regeneration in M. urundeuva, in order to increase the species multiplication. Two experiments were conducted: 1) concentrations of 6-benzylaminopurine (BAP) (0.0, 2.0, 4.0, 8.0 and 16.0 µM), in association with naphthaleneacetic acid (NAA) (0.0, 1.5 and 3.0 µM), in explants (cotyledon, hypocotyl and cotyledonary node); 2) concentrations of meta-topolin (mT) (0.0, 2.0, 4.0, 8.0, 16.0 and 32.0 µM) in explants (biaxillary, medial uniaxillary and apical basal nodal segment). The percentage of explants responsive to shoot regeneration, percentage of callus explants, number of shoots and shoot length were evaluated. In the first experiment, the shoot regeneration occurred only in explants of the cotyledonary node and hypocotyl type, with the highest responsiveness percentage (76.67 %) and number of shoots (1.97 and 1.63) obtained for the cotyledonary node in the presence of 3.0 µM of NAA in association with 2.0 (1.97 shoots/explant) and 4.0 µM (1.63 shoots/explant) of mT. In the second experiment, the resolution of the obtained quadratic equation indicates that the use of basal explant with 24.59 µM of mT added to the culture medium leads to the highest number of shoots (1.86). However, despite the mT having increased the mean number of shoots, all treatments containing this cytokinin showed callus formation. As a conclusion, it is possible to regenerate shoots in M. urundeuva from the cotyledonary node using BAP in association with NAA.

In vitro morphogenesis of Physalis ixocarpa Brot ex. Horm

ABSTRACT Physalis ixocarpa Brot. ex Horm. is considered the most economically important species of the genus. Tissue culture is pointed out as a strategy for its propagation, but researches indicate that in vitro responses are genotype-dependent. This study aimed to evaluate the in vitro morphogenesis of the P. ixocarpa green and purple varieties, in view of the massive propagation of the species. The morphogenic capacity of the explants cotyledonary node, hypocotyl and cotyledon was evaluated in Murashige & Skoog medium supplemented with benzylaminopurine - BAP (0.00, 2.5, 5.0, 7.5 or 10.0 μM) and naphthaleneacetic acid - NAA (0.00 or 2.5 μM), using a completely randomized experimental design, in a 3 x 5 x 2 factorial scheme, with 30 treatments for each variety. The number of shoots per direct and indirect organogenesis and the percentage of explants with callus were analyzed. The in vitro morphogenetic expression of P. ixocarpa is influenced by the type of explant and by the plant regulators BAP and NAA. The cotyledonary node explant is efficient for the production of shoots via direct organogenesis in the two varieties studied.

In Vitro Regeneration Potential of White Lupin (Lupinus albus) from Cotyledonary Nodes

The tissue culture regeneration system of Lupinus albus has always been considered as recalcitrant material due to its genotype-dependent response and low regeneration efficiency that hamper the use of genetic engineering. Establishment of repeatable plant regeneration protocol is a prerequisite tool for successful application of genetic engineering. This aim of this study was to develop standardized, efficient protocol for successful shoot induction from cotyledonary node of white lupin. In this study, 5 day old aseptically cultured seedlings were used to prepare three explants (half cotyledonary node, HCN; whole cotyledonary node, WCN; and traditional cotyledonary node, TCN), cultured on four concentrations of M519 medium (M519, ½ M519, 1/3 M519, and ¼ M519), containing four carbohydrate sources (sucrose, fructose, maltose, and glucose), and stimulated with various combinations of KT (kinetin), and NAA (naphthalene acetic acid) for direct shoot regeneration. High frequency of 80% shoot regeneration was obtained on ½ M519 medium (KT 4.0 mg L−1 + NAA 0.1 mg L−1) by using HCN as an explant. Interestingly, combinations of (KT 4.0 mg L−1 + NAA 0.1 mg L−1 + BAP 1.67 mg L−1), and (KT 2.0 mg L−1 + NAA 0.1 mg L−1) showed similar shoot regeneration frequency of 60%. Augmentation of 0.25 g L−1 activated charcoal (AC) not only reduced browning effect but also improved shoot elongation. Among the all carbohydrate sources, sucrose showed the highest regeneration frequency with HCN. Additionally, 80% rooting frequency was recorded on ½ M519 containing IAA 1.0 mg L−1 + KT 0.1 mg L−1 (indole acetic acid) after 28 days of culturing. The present study describes establishment of an efficient and successful protocol for direct plant regeneration of white lupin from different cotyledonary nodes.