Herbertia 47(1):51-52 (1991)
Propagation of Hippeastrum from floral tissues by in vitro culture
E.N. O'Rourke, W.M. Fountain and S. Sharghi

Horticulture Department Louisiana Agricultural Center
Baton Rouge, LA 70803

In 1977 and 1978 Hippeastrum hybridum floral tissues were examined for their ability to produce plantlets from calli or direct organogenesis from various regions of the inflorescence in aseptic culture. The use of floral tissues offers several advantages over other sources: it does not require the destruction of the bulb; explant material may be obtained at the time of flowering, ensuring propagation of material true-to-type; desirable seedlings may be propagated as soon as selected; above-ground parts are relatively free of soil-borne contaminants (2,4).

Hippeastrum hybridum 'Apple Blossom' flowers were excised from mature bulbs at two stages: in December and in February following natural cooling of the bulbs outdoors. Bulbs had accumulated about 280 hours of chilling below 7°C in December, while those in February had undergone about 870 hours of chilling. Floral scapes from the bulbs cut in December were about 2.3cm in length, with a range of 1.2 to 2.6cm. Scapes from February cuttage ranged from 4.7 to 7.7cm in length, with a mean of 6.0cm.

Scapes were surface-sterilized in a 0.5% solution of sodium hypochlorite in water with 2 drops of "Tween 20" surfactant per 100ml. After 10 minutes, scapes were rinsed 3 times with sterile, distilled water. Agar-based media were used in 25 x 150mm pyrex tubes and cultures were given 16 hours of light and 8 hours of darkness each day. Cool white fluorescent tubes provided a radiant flux of 94 microeinsteins m-2sec-1, and temperatures were kept at 21°C.

Petal, anther, filament, style, pedicel, and ovary tissues were excised from the surface-sterilized flowers and placed on various media using Murashige and Skoog's high salt medium (3) as the basal medium. Growth promoting substances added included (in mg per liter): meso-inositol, 100; adenine sulfate•2H2O, 80; thiamine HCl, 0.1; pyridoxine HCl, 170; sucrose, 25,000; bacto-agar, 6,000. The pH was adjusted to 5.7 prior to autoclaving 15 minutes at 121°C. The medium was supplemented with napthaleneacetic acid (NAA) at 0, 0.5, 1.0, 5.0, or 10.0mg/l and kinetin or benzylaminopurine (BA) at 0, 0.5, 1.0, 5.0 or 10.0 mg/l in all possible combinations. Four replications were used per treatment.

Slices of petal tissue measuring 3mm wide were taken from flowers perpendicular to the long axis and place distal end down on medium. Squares of petal tissue 8 x 8mm were placed on the medium, but rolled up, causing desiccation. Anthers were removed from filaments and placed horizontally on the medium. Ovaries and pedicels were placed intact, distal side down on medium in December, but they were kept separated in the February cultures, cut across the long axis into slices about 2mm thick and placed distal side down on media.

RESULTS

Cellular enlargement of the horizontal slices of petal tissue resulted in about a three-fold size increase. Tissues remained alive for more than 9 months, but did not form calluses or piantlets. The anthers dehisced along the sutures after 10 days of culture, but pollen did not shed. Specks of yellow-green callus were produced at the ends of filament and style segments on media supplemented with NAA in the presence or absence of kinetin. Callus could not be increased even on media supplemented with 10% coconut milk. Increase of the callus was unsuccessful on media supplemented with 2,4-D, 2mg/l; kinetin, 1mg/l or coconut milk, 10% by volume, as was reported by Bapat and Narayanaswamy (1).

Pedicel and ovary explants from December flowers initiated plantlets after 32 weeks in culture. 52 plantlets were obtained from 7 original explants. Plantlets formed on the inside of the ovary wall and on the placentas. This occurred when NAA and BA concentrations were 1.0 and 10, 2.0 and 5.0, and 2.0 and 10mg per liter, respectively.

Pedicel tissues from February flowers were more productive than ovary tissues. Plantlets formed on pedicel explants from inner tissues at the proximal ends. Optimum NAA and BA concentrations were the same as for the ovary-pedicel explants.

LITERATURE CITED

  1. Bapat, V.A. and S. Narayanaswamy 1976. Growth and Organogenesis in Explanted Tissues of Amaryllis in Culture. Bulletin of the Torrey Botany Club 103 (2):53-56.
  2. Heuser, C.W. and D.A. Apps 1976. In vitro Plantlet Formation From Flower Petal Explants of Hemerocallis cv. 'Chipper Cherry'. Canadian Journal of Botany 54:616-618.
  3. Murashige, T. and F. Skoog 1962. A Revised Medium For Rapid Growth and Bio-assays With Tobacco Tissue Cultures. Physiol. Plant. 15:473-479.
  4. Ziv, M., A.H. Halevy, and R. Shilo 1970. Organs and Plantlets Regeneration of Gladiolus Through Tissue Culture. Annals of Botany 34:671-676.


Herbertia 47(1):53 (1991)
Off-type plants obtained from callus cultures derived from pedicels of Hippeastrum hybridum 'Pinksterflower'
E.N. O'Rourke

Horticulture Department
Louisiana Agricultural Center
Baton Rouge, LA 70803

Explants of cross sections of pedicels of 'Pinksterflower' Hippeastrum cultured in 1977 on Murashige and Skoog high salt medium supplemented with 2,4-D at 2ppm and kinetin at 1ppm were grown to flowering in a greenhouse during 1978 and 1979 and have flowered each year since. Two types of flowers that differ from those of the original clone were obtained — one that is essentially a much smaller version of the original and the other is completely different and about 2/3 the size of flowers of the original clone.

'Apple Blossom' cultured under the same conditions produced plants that appear identical to the clone from which the explants were taken. The illustrations show the two kinds of "off-type" flowers and a comparison with a normal size 'Apple Blossom' plant obtained from callus cultures. (The labels erroneously refer to "peduncle", but the tissues were from pedicels near the ovaries of the flowers)


Figure 1. Off-type plants of 'Pinksterflower' from pedicel tissue culture.


Figure 3. Half size plants of 'Pinksterflower' from pedicel tissue culture.


Figure 2. A normal size 'Apple Blossom' plant (left) and a dwarf 'Pinksterflower' (right), both derived from tissue culture.


Herbertia 47(1):54-55 (1991)
Rapid propagation of Hippeastrum bulblets by in vitro culture
E.N. O'Rourke, W.M. Fountain and S. Sharghi

Horticulture Department Louisiana Agricultural Center
Baton Rouge, LA 70803

Many ornamental bulbous plants in Amaryllidaceae, Iridaceae, Agavaceae and Liliaceae are slow to offset naturally in those cases where offsets occur, and some good horticultural selections rarely produce offsets (4). Many Hippeastrum species and hybrids have been reluctant to offset, and the species, especially, may be difficult to maintain outside their native environments. A method of increasing vegetative propagation is desirable and this study was aimed at that objective.

EXPERIMENT 1

In 1979 shoot apices or bulblets of Hippeastrum hybridum 'Apple Blossom' were obtained from scale-stem fractions maintained in a watersaturated atmosphere (3,5). Mature bulbs of flowering size (20 to 25 cm circumference) were the source of at least 40 fractions per bulb with bulblets averaging 2.5 per fraction after 3 weeks. The small bulblets were about 1.5 to 2mm in diameter and about 2.0 to 3.0mm long at this time. The small buiblets were excised from the scale-stem fractions, surface-sterilized by immersion for 10 minutes in a solution of 0.5% sodium hypochlorite in water with 2 drops of the surfactant "Tween 20" added per 100ml. Following immersion the bulblets were rinsed 3 times in sterile, distilled water. They were then placed on nutrient agar in 25 x 150mm pyrex tubes closed with metal closures. The tubes were maintained under cool white fluorescent lights providing a radiant flux of 94 microeinsteins m-2sec-1 for 16 hours in each 24 hour period with 8 hours of darkness. The temperature was about 21°C.

The culture medium for all experiments contained Murashige and Skoog's inorganic salts (2) and (in mg per liter): meso-inositol, 100; adenine sulfate•2H2O, 80; thiamine HCl, 0.1; pyridoxine HCl, 0.5; nicotinic acid, 0.5; glycine, 2.0; NaH2PO4•H20, 170; sucrose, 25,000; bacto-agar, 6,000. The pH was adjusted to 5.7 prior to autoclaving for 15 minutes at 121°C. Auxin supplements to the basal medium were: napthaleneacetic acid (NAA); indoleacetic acid (IAA), 4-chlorophenoxypropionic acid (4-CPA), or 2,4-dichlorophenoxyacetic acid (2,4-D) at 0, 0.50, 1.00, 2.00, or 4.00mg per liter. Cytokinins supplemented to the basal medium included the following: kinetin, 6(8,8-dimethylalylamino purine) (2iP), or 6-benzylaminopurine (BA) at 0, 0.5, 1.0, 5.0 or 10.0 mg/l. Comparisons made were NAA, kinetin; NAA, 2iP; NAA, BA; IAA, kinetin; IAA, BA; 4-CPA, BA; 2,4-D, kinetin and 2,4-D, BA. Comparisons were also made between NAA at 0, 0.5, 1.0, 2.0 or 4.0 mg/l and BA at 0, 0.5, 1.0, 5.0 or 10.0 mg/l. All possible combinations were made, using 4 replications per treatment.

EXPERIMENT 2

Bulblets 5 to 6 mm in diameter were halved or quartered longitudinally and placed on the basal medium supplemented with NAA at 0, 0.5, 1.0, 2.0 or 4.0 mg/l and BA at 0, 0.5, 1.0, 5.0 or 10.0 mg/l. All possible combinations were made, using 4 replications per treatment.

RESULTS

The addition of auxins and cytokinins to the basal medium on which entire bulblets were cultured failed to promote offset formation over 16 weeks. All bulblets developed roots and leaves in a normal manner regardless of the type or concentration of growth regulator employed. Hussey (1) reported that medium supplemented with BA at 2.0mg/l caused bulblets to produce 1 to 3 offsets.

Bulblets that were halved or quartered produced single bulblets from each fraction. The new bulblets attained the size of the original bulblet in the 10th to 12th week after cutting. Growth regulators did not produce any differences in the growth rates of the new bulblets nor in numbers produced that were significant.

Each large bulb originally cut into scale-stem fractions, with new bulblets subsequently halved or quartered produced about 100 new bulbs. The cuttage could be repeated every 10th or 12th week with a theoretical increase of in excess of 25,000 bulblets within a year after the mother bulb is cut. This would represent about a 250-fold increase over the numbers resulting from the scale-stem fraction method of cuttage.

LITERATURE CITED

  1. Hussey, G. 1976. In Vitro Release of Axillary Shoots From Apical Dominance in Monocotyledonous Plants. Annals of Botany 40:1323-1325.
  2. Murashige, T. and F. Skoog 1962. A Revised Medium For Rapid Growth and Bio-assays With Tobacco Tissue Cultures. Physiol. Plant 15:473-479.
  3. Traub, H. 1935. Propagation of Amaryllis by Stem Cuttage. Herbertia 2:123-126.
  4. Traub, H. 1958. The Amaryllis Manual. Macmillan and Co., New York.
  5. Yusof, I.B.M. 1971. Vegetative Propagation of Amaryllis (Amaryllis cv. leopoldaeoides Traub). PhD Thesis, Louisiana State University, Baton Rouge, LA.

Herbertia 47(1):58 (1991)
Some observations on increase of Hippeastrum hybridum bulbs by cuttage
E.N. O'Rourke

Horticulture Department
Louisiana Agricultural Center
Baton Rouge, LA 70803

Six bulbs, about 24-26cm in circumference, of the Hippeastrum clone 'Wedding Bells' were cut into "scale-stem fractions" according to the method of Traub (1935) and placed into fruit jars in a water-saturated atmosphere and maintained under fluorescent lights (cool white tubes providing about 150 foot-candles of illuminance at jar level) for 16 hours daily with 8 hours of darkness. The temperature was near 21°C under the lights. When bulblets developed to 14 inch in diameter, they were removed and planted in a greenhouse bed containing 3 parts sand and I part vermiculite. Scale-stem fractions were returned to the jars, where they made additional bulblets. Small bulblets were large enough to be removed about one month after they were placed into the jars in early May, 1971, and additional bulblets were removed and planted in the greenhouse until September, 1971. Bulbs were grown and fertilized intermittently with soluble fertilizer at about 300ppm concentrations with equal parts of N, P, and K. Fertilizer was applied every fourth watering. In October, 1972, plants were removed from the greenhouse bed and dried. At that time the numbers and sizes of bulbs were recorded. The older bulbs were about 16 months old at the time of the counting and the youngest about 13 months old.

Hippeastrum hybridum 'Wedding Bells' bulbs from cuttage
Size (Inch diameter)
Number of Bulbs
3 or over (These show flower scapes) 5
2 3/4 2
2 10
13/4 5
1 1/2 - 13/4 10
1 1/4 - 1 1/2 18
1 - 1 1/4 30
1 23
3/4
10
1/2
8
3/8
1
Total 122
Average per bulb 20.3

LITERATURE CITED