Yukiko Shimada (The Hiroshima Botanical Garden)
Propagation by leaf piece cutting of Begonia Tuberous Group

 

Propagation by leaf piece cutting of Begonia Tuberous Group

Begonia Tuberhybrida Group has large, flattish perennial tubers. Their ancestors are seven tuberous species that inhabit the Andes of South America. Their various plant forms, flower sizes and colors make them one of the most popular pot flower crops.

  Rhizomatous begonia plants including the Rex-cultorum Group, and B.~ cheimantha Everett ex. C. Weber plants form adventitious buds on leaf cutting. However, the B. Tuberhybrida Group is propagated mainly by seeds, because vegetative propagation with cutting has been considered impossible. However, seedlings generally exhibit a wide range of variations because of their high levels of heterozygosity and their limited pollination period. Therefore, the establishment of a vegetative propagation method for plants with excellent horticultural characteristics is in demand. Although most of the research on vegetative propagation of B. Tuberhybrida Group has been on in vitro propagation, the systems developed are not practical because of the presence of endotrophic bacteria in the tissues.

The aim of the present work was to study the conditions suitable for promoting adventitious bud formation in leaf piece cutting of B. Tuberhybrida Group, and to establish a new and efficient method of vegetative propagation.

 

Chapter 1. Frequency of adventitious bud formation in tuberous and other species

Whole leaf blades of four tuberous and 15 erect stemmed and 23 rhizomatous species were placed in distilled water, and the course of organogenesis was compared. Three tuberous species were the main ancestors of B. Tuberhybrida Group.

The majority of rhizomatous species formed adventitious buds on the cut end of the leaf blade, while hardly any  of the tuberous and erect stemmed species formed them. Thus the lack of ability for adventitious bud formation in the ancestors of the B. Tuberhybrida Group can be assumed to be the reason why propagation by leaf cutting of the B. Tuberhybrida Group has generally been considered to be impossible.

There was no correlation between frequency of adventitious bud formation and continent of origin, size of leaf blade, thickness of leaf blade, or width of a main vein. There was a tendency for the percentage of adventitious bud formation of species having palmate veins to be higher than for those having pinnate veins. Species having some multiseriate hairs also showed higher percentages of bud formation than those which have no hairs or uniseriate hairs.

 

Chapter 2. Factors promoting adventitious bud formation of B. Tuberhybrida Group

Expanded young leaves of B. Tuberhybrida Group were detached from the stock plants and cut radially into four pieces from the base. Each leaf piece was inserted into one of five media: perlite, Kanuma soil, vermiculite, rockwool granule (rock fiber) or rockwool blocks  in nursery trays. Only 20% or fewer of the leaf pieces that were inserted in perlite, Kanuma soil, vermiculite and rock fiber formed adventitious buds, whereas 50% of those inserted in the rockwool blocks formed adventitious buds.  The rockwool blocks were therefore used in subsequent work.

Leaf pieces were inserted into rockwool blocks on 8 April, 20 May, 29 July and 13 October. The percentages of adventitious bud formation were 67% in April and 60% in October, whereas in July only 13% formed adventitious buds. Next, leaf pieces were inserted in rockwool blocks kept at 15, 20, 25, or 30‹C under plant environmental control systems. The percentages of adventitious buds formed were higher at 15 and 20‹C than at 25 and 30‹C. Therefore, it appears  that the optimum temperature for adventitious bud formation is around 15-20‹C.

The effects of size and configuration of leaf cuttings on adventitious bud formation was determined. Leaf pieces of B. Tuberhybrida Group eTenellaf were used for the experiment and five cutting configurations were compared.  They were: whole leaf blade with 5 mm petiole, 5 ~ 4 cm leaf piece with 5 mm petiole, 2 ~ 1.5 cm leaf piece with 5 mm petiole, 2 ~ 1.5 cm leaf piece cut at leaf base without petiole, 2 ~ 1.5 cm leaf piece cut at 2 cm further away from a leaf base. The different types of leaf pieces were excised and inserted in rockwool blocks containing distilled water. Seventy three percentages of whole leaf blades with petioles formed adventitious buds, whereas smaller leaf pieces did not form adventitious buds at all and instead formed adventitious roots or calli.

2 ~ 1.5 cm leaf pieces with 5 mm petioles of  five cultivars of B. Tuberhybrida Group were inserted in rockwool blocks containing distilled water. None of the cultivars  formed any adventitious buds.

 

Chapter 3. Effects of plant growth regulators on adventitious bud formation in leaf piece cutting

It was anticipated that adventitious bud formation in 2 ~ 1.5 cm leaf pieces might be induced with high frequency by applying plant growth regulators. The leaf pieces were inserted in rockwool blocks containing different concentrations (0, 0.01, 0.1, 0.25, or 0.5 ppm) of NAA (ƒ¿-naphthalene acetic acid) alone or (0, 0.05, 0.1, 0.25, 0.5, 1, or 2 ppm) of BA (6-benzylaminopurine) alone. About 20% of the leaf pieces inserted in the absence of NAA and BA became brown and died, the surviving leaf pieces did not form any adventitious buds at all. All of the leaf pieces treated with more than 0.1 ppm NAA died after showing leaf yellowing. All leaf pieces with more than 0.25 ppm BA survived without browning and/or yellowing and 80% of them formed adventitious buds. The surviving percentage was high with 0.1-0.5 ppm NAA plus 0.5 ppm BA, and the pieces did not turn yellow, but callus formation was promoted and the onset of adventitious bud formation was delayed. 

Changes in the chlorophyll contents in leaf pieces inserted in rockwool blocks containing 0.5 ppm NAA, 0.5 ppm BA or free of growth regulators were investigated. After 10 days from inserting, total chlorophyll contents in leaf pieces treated with 0.5 ppm NAA decreased, and then the leaf began to yellow a little. Treatment of BA inhibited chlorophyll degradation in leaf pieces. Ethylene production from leaf pieces stayed low throughout the culture period, whether leaf pieces were treated with NAA and BA or not. Therefore, chlorophyll degradation in leaf pieces treated with NAA was not related to ethylene production.

Changes in total polyphenol contents in leaf pieces were investigated in comparison with BA treatment or in its absence. Total polyphenol contents in both sets of leaf pieces increased gradually after cutting: the amount of increase in non BA-treated leaf pieces was significantly more than that of BA-treated, and non BA-treated leaf pieces turned brown. Therefore, it was concluded that BA treatment reduced the rate of increase of total polyphenol contents and inhibited browning of leaf pieces.  It was therefore adopted for the later studies.

 

Chapter 4.  Effects of day length during growth of mother plant and leaf piece cutting, section position and orientation of leaf piece on adventitious bud formation

Mother plants were grown under 18 hr day length prior to this experiment. On 11 October, the mother plants were placed in a glasshouse with a night temp 14‹C@under 18 hr day length (LD) and natural day length (SD). On 23 November, young, but fully opened leaves were selected for cuttings and were cut to 2 ~ 1.5 cm leaf pieces. The leaf pieces were inserted in rockwool blocks containing 0.5 ppm BA, then they were maintained in a glasshouse with a night temp 14‹C under 18 hr and natural day length. Almost all of the leaf pieces from the LD mother plants formed adventitious buds, no matter what was the cuttingsf day length regime, whereas 60-67% of leaf pieces taken from SD mother plants survived, but their percentage of adventitious bud formation was only 13%. The LD mother plant and LD cutting combination hastened the development of adventitious buds.

2 ~ 1.5 cm leaf pieces were cut at 0, 1, 1.5 or 2 cm away from the base towards the tip of leaf blades and inserted in rockwool blocks containing 0.5 ppm BA. The percentage of adventitious bud formation declined from the base towards the tip of the leaf, that is, the leaf pieces cut at 0 cm from the leaf base formed adventitious buds at the highest rate (87%), followed by 1 cm (33%), 1.5 cm (40%) and 2 cm (0%). The greater part of the leaf pieces cut further away from the base formed calli.

The leaf pieces cut at 2 cm from the leaf base were placed vertically upright, horizontally or vertically inverted in rockwool blocks containing 0.5 ppm of BA. When leaf pieces were placed vertically upright, none formed adventitious buds and most formed calli. However, when they were placed horizontally or vertically inverted, the percentages of adventitious bud formation were 60% and 80%, respectively. The leaf pieces placed vertically inverted formed small bud primordia directly without callus on some lateral veins of the distal cut end (the end immersed in the medium). When the leaf pieces placed vertically were inverted in rockwool blocks containing 0.25 ppm of NAA and 0.5 ppm of BA, none formed adventitious buds and all formed calli. In addition, when leaf pieces to which lanolin paste containing 100 ppm TIBA (2,3,5-triiodobenzoic acid) had been applied were inserted vertically upright in rockwool blocks containing 0.5 ppm BA, 73% of them formed adventitious buds. This can be attributed to the disturbance of the basipetal movement of auxin by inversion. Based on these results, it appears that a suitable balance of endogenous auxin and endogenous cytokinin may be established near this distal cut end with assisting of cytokinin exogenously supplied as a plant growth regulator, BA, too, and the resulting in the formation of adventitious bud primordia.

In order to know whether adventitious buds obtained by this leaf piece cutting method arose directly from the original tissues or indirectly through a callus phase, adventitious bud histogenesis was investigated. Adventitious primordia differentiated directly from epidermal and subepidermal layers. Ten plants propagated by this method showed no morphological aberrants for flower and leaf characteristics in comparison with the mother plant. Additionally, the chromosome numbers of both mother plant and plants propagated were the same (2n=28) and their karyotypes at metaphase were similar to each other. Therefore, it appeared that there were no chromosomal aberrations.

Finally, various begonias for which percentages of adventitious bud formation were low in chapter 1 were tried out again using the newly developed technique. 2 ~ 1.5 cm leaf pieces cut at the leaf bases of the begonias were inserted vertically upright in rockwool bed containing 0.5 ppm BA. Many species and cultivars except for two erect stemmed begonias formed adventitious buds. When leaf pieces of those two eresistantf begonias were inserted vertically inverted in the medium, the percentages of adventitious bud formation were 93% and 73%.

 In conclusion, vegetative propagation by the use of leaf piece cutting of Begonia Tuberhybrida Group, which had been considered almost impossible until now, was successfully developed. Rockwool blocks provided a suitable bedding medium and the optimum temperature for adventitious bud formation was around 15-20‹C. It was found that 0.5 ppm BA enhanced survival, inhibited browning, and promoted adventitious bud formation of small leaf pieces. The frequency of bud formation was highly dependent on the position of the pieces in the leaf blade, declining from a high rate at the base towards no bud formation at the tip. Adventitious bud formation on pieces without the leaf bases was achieved by placing them vertically inverted, or by inserting them vertically upright with the addition of an application of lanolin paste containing 100 ppm TIBA. Plants propagated by this cutting method were not aberrant. Moreover, this method brought good results for other tuberous and erect stemmed begonias.

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