Propagation of Aloe polyphylla Tissue Culture vs. Seed

Alan C Beverly - Spring 2023

The reproduction of Aloe polyphylla, an endangered species I introduced to the nursery industry back in 1991, is difficult because in the genus Aloe you must have a stem section to root. For this basal rosette form there is effectively no stem, there is no meristem on any leaf which can be coaxed into developing. You must have seed . For A.p. only adult plants (> 150 leaf) will flower in the Spring, and may not repeat again the next year. This makes seed rather precious. In the first 35 years of my involvement with A.p. I was not able to produce much seed, and many germination trials gave poor results. I have since been able to breed hybrid seed by the thousands because I had mature plants to create hybrids, and found the correct seed germination formula. The pollination biology favors outcrossing , but selfed seed is viable.

Nurserymen, wowed by the architectural form of this species, were frustrated by this propagation challenge and turned to tissue culture for results which I show here are disappointing . The science of tissue culture is well understood, worked out by Abrie & Van Staden (1) , and the nursery industry has successfully applied this technique to many species in their quest to produce plants which could drive sales. Nobody suspected that the results I reveal here for A.p. would be so disappointing.

The first 7 years of A.p. TC ( tissue culture) plants’ life are nothing exciting because the visual sense of 5 spiral rows of sequenced leaf is confused and disorganized and depends on the plant reaching the ~45 leaf stage. These TC plants only reward a new owner with a visual spiral pattern in the 8th year. These plants require more development time than a seed grown plant ,which shows spiral growth pattern in the second year. I can raise a 65 leaf plant from seed in two years and it shows either a Right, or Left spiral orientation. Photo A shows a correct sequence on an adult (~150 leaf) plant.

Clones obtained from a local grower were cultivated for several years and planted into the landscape in a sandy, well drained soil. The photos B,C,D show plants about 15” diameter and about the 75+ leaf stage with abnormal, confused leaf sequence difficult to describe. These plants have not been able to form spirals we witness in seed grown plants which divide in a specific way in space and time to produce the spiral sequence normal to seed grown plants. A seed embryo comes equipped and organized to produce the recognizable pattern on day one of its life. I have seen wild plants and in my nursery change growth orientation from R to L ,and L to R . They look strange , but are not confused. There is thus NO genetic prescription to the growth orientation of any plant . New leaf emerge from the center as older leaf , appressed to the soil and shaded , are resorbed . In this way the plant continuously re-invents itself.The service life of a single leaf in an adult plant (~150 leaf) is 2-3 years . The developmental dynamics of the apical meristem cell are fickle, and vulnerable to perturbations . I never saw any confused leaf sequences in any wild plants, nor in any of my nursery plants. An adult plant will sometimes undergo binary fission, which then requires 2-3 years for the two plants (identical genotypes) to grow apart. I have witnessed this many times in nursery plants. Sometimes small “pup” plants will emerge from below . Not enough to use for propagation.

Spiral phyllotaxy is common in the plant kingdom. There are invertebrates species that also find the Fibonacci series a solution to their body plan. The explanation is still held secret by an embryo cell , and the exacting sequence of events and how the controls work remains a mystery to biologists. Siobhan and Kuhlemeier (2) reviewed how plants develop leaf and have offered insight into the A.p. clone dilemma. The first cell division of an embryo in a germinating seed established root-shoot polarity. Then subsequent cell divisions establish the proto-tissue of each. Differentiation of cells begins to designate their future function. A general principle in botany is that all plant cells are totipotent, that any cell has the ability to develop into another whole plant. In tissue culture cells are not organized as in a seed embryo, and are slower to develop. That TC plants still eventually develop into whole plants is one of biology’s unsolved mysteries.

“How this early embryo forms itself into an organized entity has long been considered by many to be taxonomically significant “ (Lersten, 3)

Many researchers have wondered how to discover the answers but there is little to report. We know that genes are a blueprint for synthesis of specific proteins , but do not act as directors of organogenesis. It is achieved by macromolecular self assembly from the bottom up.as opposed to from the top down. This gap in embryology is so profound that Dawkins (4) has underscored it in a discussion of how organogenesis occurs. Siobhan & Kuhlemeier (2) discuss gradients of hormone signals in varying ratios to accomplish specific cell function and leaf development . Other researchers have focused on using genetic mutants (5,6) to learn how it happens in Arabidopsis.

Whatever plant biologists decide about organogenesis I am sure that seed grown A.p. have an advantage over TC plants . I pledge to continue the effort to produce genetic recombinants to help save this wonderful, amazing endangered species

Photo A: A Seed grown adult plant showing a R hand spiral growth

Photo B : A ~5 year old TC plant with confused leaf sequence

Photo C: A TC plant with confused leaf sequence

Photo D : A TC plant with confused leaf sequence

References:

1. Abrie,A.L. & Van Staden Jan 2001 Plant Growth Regulation 33 (1):19-23 “Micropropagation of the Endangered Aloe polyphylla”

2. Siobhan,A. & Cris Kuhlemeier 2010 The Plant Cell vol 22 : 1006-1018 “How A Plant Builds Leaves”

3. Lersten, Nels Flowering Plant Embryology Blackwell 2004

4. Dawkins , Richard The Greatest Show On Earth pg214-218

5. S. Shannon & D.Ry Meeks-Wahner 1991 The Plant Cell vol 3: 877-892 “A Mutation In The Arabidopsis Gene TFL 1 Affects Inflorescence Development”

6. S. Shannon & D.Ry Meeks-Wagner 1993 The Plant Cell vol639-655 “ Genetic Interactions That Regulate Inflorescence Development In Arabidopsis “