K.K. Kartha

Callus formation and plant regeneration from mesophyll protoplasts of rape plants (Brassica napus L. cv. Zephyr)

Abstract Protoplasts from mesophyll cells of rape plants ( Brassica napus L. cv. Zephyr) were isolated by enzymatic removal of cell walls in an osmoticum consisting of sorbitol and mannitol. A two-step enzyme treatment involving 0.5% each of Onozuka P1500 cellulase and Rhozyme HP150 hemicellulase followed by 0.5% each of Driselase and hemicellulase at pH 6.2 resulted in the production of viable protoplasts in 80–90% yield. The protoplasts were cultured in droplets of B5 medium in petri dishes at a light intensity of 100 lux and 26°. They were able to regenerate cell walls. Cell division was apparent after 30 h of culturing and by 60 h up to 38% of the protoplasts had divided once. Cell clusters were formed within 15 days of culturing. The calli originating from cell clusters were induced to differentiate and to form complete plants.

Regeneration of cassava plants from apical meristems

Abstract Cassava plants were regenerated from meristems of five cultivars; Colombia No. 800, Llanera, Venezuela No. 255, Ecuador No. 133 and Mexico No. 35. Using benzyladenine (BA), gibberellic acid (GA3) and naphthaleneacetic acid (NAA) at molar concentrations of 5·.10−7, 10−7 and 10−7, Respectively, resulted in complete plant development on Murashige-Skoog (MS) medium supplemented with vitamins as in B5. GA3 in combination with NAA resulted in root formation, while BA with NAA produced callus and storage roots. The meristems were cultured in a growth cabinet at 26°, 60% relative humidity, and exposed to a light intensity of 4000 lux from cool, white fluorescent lamps using a light and dark cycle of 18/6 h.

Freeze-preservation of pea meristems in liquid nitrogen and subsequent plant regeneration

Abstract A procedure has been developed for the freeze-preservation of pea ( Pisum sativum L.) meristems and subsequent plant regeneration. The meristems were precultured in the presence of 5% DMSO for 48 h followed by freezing at cooling velocities varying from 0.5 to 1.0°C/min up to a terminal freezing temperature of −40°C and subsequently stored in liquid nitrogen (−196°C). Maximum survival as determined by the regrowth of meristems into whole plants was obtained at a cooling rate of 0.6°C/min after 1 h storage in liquid nitrogen (73%). The meristems were in storage in liquid nitrogen for up to 26 weeks and upon reculture more than 60% of the meristems differentiated into whole plants. Cold conditioning of meristems at 4°C prior to freezing without preculture in the presence of DMSO resulted in lower survival rates. Glycerol and ethylene glycol were ineffective cryoprotectants. Ultra-rapid freezing of meristems by direct immersion in liquid nitrogen was also lethal.

Morphogenetic Investigations on in vitro Leaf Culture of Tomato (Lycopersicon esculentum Mill. cv. Starfire) and high Frequency Plant Regeneration

Summary Leaf explants of tomato ( Lycopersicon esculentum Mill . cv. Starfire ) were cultured in vitro on Murashige and Skoog medium under controlled environmental conditions. Growth and morphogenetic responses of the explants to cytokinins and auxins were compared at different concentrations and combinations. Combinations of benzyladenine (BA) and indoleacetic acid (IAA) were more effective in inducing shoot differentiation than those of BA and naphthaleneacetic acid (NAA). Multiple shoot differentiation on the calli occurred at 10 μM BA with various concentrations of IAA. Rooting of the differentiated shoots was readily achieved by culturing the shoots on medium containing no growth hormones. Kinetin (K) in conjunction with IAA or NAA failed to induce shoot differentiation. Shoot and root differentiation also occurred at 0.1 μM zeatin (Z) + 10 μM IAA or 1 μM Z + 0.1 μM IAA. When used alone, BA or Z at 5 μM induced complete plant regeneration from the callus. Large numbers of regenerated plantlets were transferred to pots and grown to mature plants.

Regeneration of pea (Pisum sativum L.) plants from shoot apical meristems

Summary A procedure has been developed to obtain complete plants from meristems of three cultivars of Pisum sativum L., namely «Century», «Laxtons Progress» and «Afghanistan». Benzyladenine (BA) alone or in combination with naphthaleneacetic acid (NAA) at molar concentrations of 5 × 10 −7 and 10 −6 , respectively, induced shoot differentiation in meristems cultured on B5 medium at 26° C, 60 % relative humidity and exposed to fluorescent light (4000 lux, photoperiod 18 hrs). When applied alone, NAA induced complete plant formation. Root formation, on the shoots produced by culturing meristems, was induced by reculturing the shoots, 2 cm long, on half strength B5 medium supplemented with NAA at a concentration of 10 −6 M.

Factors affecting the isolation and callus formation in protoplasts from the shoot apices of Pisum sativum L

Abstract Protoplasts were isolated from shoot tips of 3-day-old seedlings of Pisum sativum L. var. Century by enzymatic degradation of the cell walls. The protoplasts originated from cells of the meristematic domes and parenchymatous tissues of 2- 3-mm tip sections using two consecutive steps of enzyme treatment in the presence of hexitols and calcium salts. Upon culturing, wall regeneration had occurred after 24 h, and cell division was observed within 3–5 days. Supplementing the culture medium with 2−5 mM l -glutamine, 5−10 μM kinetin, 1−5 μM dichlorophenoxyacetic acid (2,4-D) and 0.1−0.5 μM naphthaleneacetic acid (NAA) resulted in sustained cell division and callus formation in five weeks. Glucose at 0.3 M was superior to sorbitol and mannitol as osmotic stabilizers.

Germplasm preservation of coffee (Coffea arabica L.) by in vitro culture of shoot apical meristems

Abstract A technique for the germplasm preservation of coffee (Coffee arabica L.) by the culture in vitro of shoot apical meristems is described. Growth responses in vitro of the Colombian cultivar ‘Caturra Rojo’ and the Brazilian cultivar ‘Catuai’ were identical. Multiple shoot regeneration occurred when the meristems were cultured on Murashige and Skoog (MS) medium supplemented with 5 and 10 μM of either benzyladenine (BA) or zeatin or in combination with 1 μM napthalene acid (NAA) while single shoots were regenerated at lower levels of cytokinins. Root regenerarion occurred in high frequency only when the differentiated shoots were recultured on sucrose-free 1 2 strength MS medium supplemented with 1 μM indolebutyric acid (IBA). The regenerated plantlets were maintained in vitro at 26°C for over 2 years without periodic transfer and some of them were successfully grown in pots. The application of this technique to the germplasm preservation of coffee is discussed.

Regeneration of plants from callus tissue of the forage legume Stylosanthes guianensis

Abstract Plants were regenerated from leaflets of Stylosanthes guianensis (Aubl.) Sw. cultured in vitro. Explants from both mature and immature leaflets were induced to form callus when aseptically cultured on Murashige and Skoog salts containing vitamins as in B5 medium, 3% sucrose, 0.8% agar and three combinations of napthaleneacetic acid (NAA) and benzyladenine (BA). Regeneration of shoots was readily achieved by transferring pieces of callus, even after subculturing for 6 months, onto fresh medium containing a low NAA/BA ratio. Shoots were induced to form roots upon transfer to medium devoid of hormones.

Effect of Growth Regulators on Plant Regeneration from Shoot Apical Meristems of Cassava (Manihot esculenta Crantz) and on the Culture of Internodes in vitro

Summary The regeneration of shoots from cassava meristems was described as a function of presence or absence of cytokinins, NAA and GA 3 Out of 4 cytokinins, BA was best suited for plant regeneration followed by zeatin, kinetin and 2iP. In conjunction with either 1.0 or 0.1 µM NAA and 0.1 µM GA 3 all levels of BA induced plant regeneration in 100% of the meristems. Shoot development was poor in most combinations involving higher concentrations of NAA. Addition of GA 3 improved plant regeneration and its effect was more pronounced at higher concentrations of NAA. Attempts to induce shoot regeneration from callus derived from internodal segments were unsuccessful.

The Effect of Canavanine on the Growth of Cells from Suspension Cultures and on Intergeneric Heterokaryocytes of Canavanine Sensitive and Tolerant Plants1)1)NRCC NO. 14920

Summary Through application of the polyethylene glycol method, protoplasts of canavanine sensitive soybean cell cultures were fused with canavanine tolerant protoplasts from alfalfa, Caragana, and sweet clover. Within several days of culture the fusion products, heterokaryocytes, divided and developed into small cell clusters. The heterokaryocytes exhibited a sensitivity to canavanine as strong as soybean protoplasts. The results indicate that protoplasts from cultured soybean cells and from mature leaves of alfalfa, Caragana,and sweet clover when fused do not generate an incompatibility which would prevent cell division. The reaction pattern of the heterokaryocytes appeared to be determined by the soybean partner.